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Enzymology| Volume 285, ISSUE 30, P22986-22998, July 23, 2010
Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease*
Yasuko Ono
From the Calpain Project, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 156-8506, Japan,
Koichi Ojima
CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan, and
Fukuyo Torii
the Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
Emi Takaya
Naoko Doi
Kazuhiro Nakagawa
Shoji Hata
Keiko Abe
Hiroyuki Sorimachi
To whom correspondence should be addressed: Calpain Project, Rinshoken, Kamikitazawa 2-1-6, Setagaya-ku, Tokyo 156-8506, Japan. Fax: 81-3-5316-3163;
Show footnotesHide footnotes
Author Footnotes
* This work was supported in part by MEXT.KAKENHI Grants 18076007 (to H. S.) and 22770139 (to Y. O.) and JSPS.KAKENHI Grants 18700392 and 20500369 (to K. O.) and 19658057 and 20370055 (to H. S.), by Research Grant 20B-13 for Nervous and Mental Disorders from the Ministry of Health, Labour, and Welfare (to H. S.), and by a Takeda Science Foundation research grant (to H. S.).
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2, Figs. S1–S5, and Videos S1 and S2.
1 These authors contributed equally to this work.
2 Present address: Meat Proteins Research Team, NILGS, Tsukuba 305-0901, Japan.
3 Present address: Life Science Laboratory, Advanced Materials Laboratories, Sony Corporation, Yushima, Tokyo 113-8510, Japan.
Open AccessPublished:May 11, 2010DOI:https://doi.org/10.1074/jbc.M110.126946
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Because intracellular [Na+] is kept low by Na+/K+-ATPase, Na+ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca2+-activated intracellular "modulator proteases" that is responsible for a limb-girdle muscular dystrophy ("calpainopathy"), underwent Na+-dependent, but not Cs+-dependent, autolysis in the absence of Ca2+. Furthermore, Na+ and Ca2+ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na+/K+-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na+/Ca2+-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca2+-binding sequence in the protease acted as a Na+ sensor. Proteomic analyses using Cs+/Mg2+ and p94CS-KI mice as negative controls revealed that Na+ and Ca2+ direct p94 to proteolyze different substrates. We propose three roles for Na+ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca2+], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na+-dependent enzyme.
Proteolytic Enzymes
Sodium Ion
Na+ is essential for all life. Its extracellular concentration is relatively high (∼145 mm), and a number of enzymes, such as blood coagulation proteases, are regulated by the [Na+] (
Dang Q.D.
Di Cera E.
Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 10653-10656
Silva Jr., F.P.
Antunes O.A.
de Alencastro R.B.
De Simone S.G.
Biophys. Chem. 2006; 119: 282-294
Kamata K.
Kawamoto H.
Honma T.
Iwama T.
Kim S.H.
Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6630-6635
). The relative intra- and extracellular concentrations of Na+, K+, and Cl− constitute the basis of the membrane potential, which is especially important for excitatory cells such as muscle cells (
Goldin A.L.
Annu. Rev. Physiol. 2001; 63: 871-894
Krishnamurthy H.
Piscitelli C.L.
Gouaux E.
). Depolarization of the muscle cell membrane caused by the opening of acetylcholine receptors results in the cell excitation-coupled contraction. Intracellular [Na+] ([Na+]i) is maintained at rather low levels (∼15 mm). To achieve this, cells express Na+/K+-ATPases on their cytoplasmic membrane and pump Na+ out of the cell in exchange for K+ (
Ogawa H.
Shinoda T.
Cornelius F.
Toyoshima C.
Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13742-13747
). Therefore, it has been believed that all Na+-dependent enzymes are extracellular.
Ca2+, like Na+, is essential for life, and the calpains constitute a superfamily of Ca2+-activated intracellular modulator proteases that regulate various cellular functions, such as signal transduction, autophagy, and apoptosis (
Goll D.E.
Thompson V.F.
Wei W.
Cong J.
Physiol. Rev. 2003; 83: 731-801
Beckmann J.S.
Spencer M.
Neuromuscul. Disord. 2008; 18: 913-921
Yousefi S.
Perozzo R.
Schmid I.
Ziemiecki A.
Schaffner T.
Scapozza L.
Brunner T.
Simon H.U.
Nat. Cell Biol. 2006; 8: 1124-1132
Suzuki K.
Hata S.
Kawabata Y.
Sorimachi H.
Diabetes. 2004; 53: S12-S18
). However, the most abundant and ubiquitous calpains (μ-calpain and m-calpain, the "conventional" calpains) require Ca2+ concentrations that are much higher (10 μm ∼ 1 mm) than the physiological intracellular [Ca2+] ([Ca2+]i, ∼100 nm) making them almost inactive in the cytosol (
). On the other hand, skeletal muscle-specific calpain, p94/calpain 3 (
Imajoh-Ohmi S.
Emori Y.
Kawasaki H.
Ohno S.
Minami Y.
J. Biol. Chem. 1989; 264: 20106-20111
Broux O.
Allamand V.
Fougerousse F.
Chiannilkulchai N.
Bourg N.
Brenguier L.
Devaud C.
Pasturaud P.
Roudaut C.
Hillaire D.
Passos-Bueno M.R.
Zatz M.
Tischfield J.A.
Fardeau M.
E. J.C.
Cohen D.
Cell. 1995; 81: 27-40
), is unique in that when generated by in vitro translation, it undergoes very rapid (t½ < 10 min) autolytic degradation even in the absence of Ca2+ (
Toyama-Sorimachi N.
Saido T.C.
Sugita H.
Miyasaka M.
Arahata K.
Ishiura S.
). When expressed in cultured cells, such as COS7 cells, p94, but not an active site-inactive mutant (p94:C129S), also autolyses extensively, without stimulation, almost disappearing from the cell (
). These findings indicate that the physiological intracellular ionic environment is sufficient for p94 to be activated, unlike conventional calpains. It should be noted, however, that rapid autolytic activity of p94 is suppressed in skeletal muscle cells in vivo, probably by its binding to connectin/titin, a gigantic elastic protein connecting the Z-band and the M-line (
Labeit S.
Kolmerer B.
Science. 1995; 270: 293-296
Ono Y.
Torii F.
Ojima K.
Doi N.
Yoshioka K.
Labeit D.
Abe K.
Maeda T.
p94 and the conventional calpains are expressed in skeletal muscle. However, the fact that p94 is a product of the gene, CAPN3, responsible for limb-girdle muscular dystrophy type 2A (also called calpainopathy) (
Marchand S.
Baghdiguian S.
Herasse M.
Stockholm D.
Suel L.
Lefranc G.
Sébille A.
J. Cell Biol. 2000; 151: 1583-1590
Kramerova I.
Kudryashova E.
Tidball J.G.
Spencer M.J.
Hum. Mol. Genet. 2004; 13: 1373-1388
) clearly indicates that p94 plays roles that are distinct from those of the conventional calpains but more critical for skeletal muscle function. One intriguing feature of calpainopathy is that pathogenic missense mutations have been located along the entire p94 molecule (
Saenz A.
Pogue R.
Grimbergen J.E.
Anderson L.V.
Beley C.
Cobo A.M.
de Diego C.
Eymard B.
Gallano P.
Ginjaar H.B.
Lasa A.
Pollitt C.
Topaloglu H.
Urtizberea J.A.
de Visser M.
van der Kooi A.
Bushby K.
Bakker E.
Lopez de Munain A.
Am. J. Hum. Genet. 1999; 64: 1524-1540
Sáenz A.
Leturcq F.
Poza J.J.
Ferrer X.
Otaegui D.
Camaño P.
Urtasun M.
Vílchez J.
Gutiérrez-Rivas E.
Emparanza J.
Merlini L.
Paisán C.
Goicoechea M.
Blázquez L.
Lochmuller H.
Walter M.
Bonnemann C.
Figarella-Branger D.
Kaplan J.C.
Martí-Massó J.F.
López de Munain A.
Brain. 2005; 128: 732-742
) (see supplemental Fig. S1), which makes it difficult to explain what kind of defects in this protease lead to calpainopathy. We have shown that the impairment of proteolytic activity of p94 by gene mutations is an essential cause of calpainopathy (
Shimada H.
). Thus, unique autolytic activity of p94 must be related to the pathogenic mechanisms of calpainopathy. However, the nature of this relationship remains elusive, especially at the molecular level.
One of the largest stumbling blocks for the analysis of p94 is that its rapid autolysis makes biochemical experiments almost impossible to conduct. To address the relevance of autolysis of p94 to the pathogenesis of calpainopathy, we used an activity-attenuated mutant, p94:N358D, in which one of the active-site residues, Asn-358, is replaced by Asp. In contrast to wild-type (WT)
The abbreviations used are: WT
wild type
[Ca2+]i
intracellular [Ca2+]
CBS-IIa
Ca2+-binding site in dIIa
CBS-IIb
Ca2+-binding site in dIIb
domain I–VI
FXa
factor Xa
IS1/2
insertion sequence 1/2
[Na+]i
intracellular [Na+]
p94CS-KI mouse
p94:C129S knock-in mouse
sarcoplasmic reticulum
supernatant
1,4-piperazinediethanesulfonic acid.
p94, which autolyses quickly in the absence of Ca2+, p94:N358D expressed in Sf-9 cells can be partially purified and, contrary to our expectation, shows a Ca2+-dependent autolysis, unlike p94:C129S (
). p94:N358D is very unstable in solutions with high salt concentrations. Therefore, we examined the relationship between autolysis and the salt concentration. We found that p94 is activated by Na+, making it the first example of an intracellular Na+-dependent enzyme. This finding helps to explain some of unique features of p94 and calpainopathy. This report characterizes Na+ dependence of p94, focusing on the possibility that the coordinated sensitivity of p94 to Na+ and Ca2+ enables its regulation to be quite sophisticated and that it governs a multiplicity of p94 functions.
EXPERIMENTAL PROCEDURES
All procedures using experimental animals were approved by the Experimental Animal Care and Use Committee of Rinshoken, and the animals were treated in accordance with the committee guidelines. C57BL/6 mice were purchased from Nihon CLEA Inc. Cre-recombinase-expressing Tg mice were provided by The Jackson Laboratory. p94CS-KI mice (Capn3C129S/C129S) were generated by introducing a missense mutation corresponding to C129S by gene targeting using 129S ES cells and the Cre-loxP system so that only mouse p94-C129S, instead of WT p94, was expressed in these mutant mice under the normal transcriptional regulation of Capn3, which will be described elsewhere.
Ojima, K., Kawabata, Y., Nakao, H., Nakao, K., Doi, N., Kitamura, F., Ono, Y., Hata, S., Suzuki, H., Kawahara, H., Bogomolovas, J., Witt, C., Ottenheijm, C., Labeit, S., Granzier, H., Toyama-Sorimachi, N., Sorimachi, M., Suzuki, K., Maeda, T., Abe, K., Aiba, A., and Sorimachi, H. (2010) J. Clin. Invest. 120, in press..
Biochemical Experiments
Pellet (Ppt) and supernatant (Sup) fractions from the gastrocnemius (GC) of WT and p94CS-KI mice were prepared as described previously (
Hayashi C.
Kitamura F.
Tagami M.
Mineki R.
Arai T.
Taguchi H.
Yanagida M.
Hirner S.
) but without adding the calpastatin peptide. The p94-N358D and p94-C129S/N358D proteins were partially purified as described previously (
). Partial purification of rat wild-type p94 was performed as follows; 150 mg of rat GC was homogenized in homogenization buffer (10 mm Tris/Cl (pH 8.0), 1 mm EDTA/KOH (pH 7.5), 1 mm dithiothreitol) containing protease inhibitors, in this case (20 μg/ml soybean trypsin inhibitor (Sigma), 28 μm E64 (Peptide Institute Inc.), 0.5 mm 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma), 0.05 mg/ml calpastatin (Takara), and 2 mm phenylmethylsulfonyl fluoride (Sigma)). The Sup obtained by centrifugation (12,500 × g, 15 min, twice) was applied to a DEAE-Toyopearl (Tosoh) column (6.3 ml) and separated into 4-ml fractions eluted by 0–0.4 m CsCl in 10 mm Tris/Cl (pH 7.5), 1 mm EDTA/KOH (pH 7.5), and 1 mm dithiothreitol (see supplemental Fig. S2). The fractions were subjected to Western blot analysis, and the fraction containing the most abundant p94 (∼0.15 m CsCl elution) was concentrated 40-fold by ultrafiltration using a Millipore Microcon YM-10 (10-kDa cut) and used for the autolysis (incubation) assay.
The autolysis assay was performed by incubating partially purified p94-N358D or p94-C129S/N358D protein (∼10 ng/μl for the 94-kDa band) (Fig. 1), the pellet and supernatant fractions from WT and p94CS-KI mouse GC (prepared as above) (Fig. 2A), a partially purified rat wild-type p94 fraction prepared as above (∼1 ng/μl) (Fig. 2B), or the lysates of the cells expressing p94 mutants (Fig. 4; see below) in 20 mm Tris/Cl (pH 7.5) and 1 mm dithiothreitol for the indicated times at the indicated temperatures with the indicated concentrations of salts (0∼300 mm NaCl, KCl, or CaCl2, or 2 mm EDTA/KOH (pH 8.0)). For CaCl2, 2× Ca2+/EGTA buffer (40 mm PIPES/KOH (pH7.2), 4 mm MgCl2, 2 mm CaCl2, and 10.0, 5.00, 2.30, 2.01, 1.95, 1.80, 1.40, and 0.00 mm EGTA/KOH (pH 7.2) for pCa = ∞, 7, 6, 5, 4.5, 4, 3.5, and 3, respectively; see Ref.
Harafuji H.
Ogawa Y.
J. Biochem. 1980; 87: 1305-1312
) was used. The intensities of the bands detected by Western analysis using the p94-specific anti-pIS2 antibody (
) were quantified by Photoshop CS2 (Adobe). The relative activities were calculated by normalization in which the background intensity and band intensity at [NaCl] = [CaCl2] = 0 (shown in Fig. 2B, lane 1) are set to 0 and 100%, respectively. The lysates of the cells expressing p94 mutants were prepared by homogenizing the cells in 20 mm Tris/Cl (pH 7.5), 1 mm EDTA/KOH (pH 8.0), and 1 mm dithiothreitol (Fig. 4).
FIGURE 1Na+-dependent autolysis of p94. A, partially purified p94:N358D protein was incubated for 0 h (lanes 1 and 7) or 15 h (lanes 2–6 and 8–12) at 4 °C at the indicated [NaCl] or [KCl]. B, p94:N358D (ND, upper) or the inactive p94:C129S/N358D double mutant (ND/CS, lower; negative control) was incubated at the indicated [CaCl2] (pCa = −log[CaCl2]) at 37 °C for 30 min. C, p94:N358D was incubated at 37 °C for the indicated times with 150 mm NaCl or 10 mm CaCl2. Note that the autolysis induced by NaCl or CaCl2 at these concentrations proceeded at similar speeds but resulted in slightly different autolytic fragment patterns. Closed and open arrowheads indicate the full-length and autolytic fragments of p94, respectively, detected by Western blotting using an anti-pIS2 antibody.
FIGURE 2Na+- and Ca2+-dependent autolysis of intact p94. A, Ppt (lanes 1–16) and Sup (17–32) fractions of skeletal muscle extracts from WT and p94CS-KI (CS) mice were incubated with 2 mm EDTA (CaCl2, −), 4 mm CaCl2 (CaCl2, +), and/or 150 mm NaCl (NaCl, +) at 37 °C for 0 (incubation, −) or 40 min (incubation, +). Asterisks denote nonspecific signals (bands around 110 and 44 kDa were derived from SERCA and actin, respectively; the three other nonspecific signals in the Sup were from the secondary antibody). B, partially purified WT p94 protein from rat skeletal muscle extract (see supplemental Fig. S2) was incubated with various combinations of NaCl and CaCl2 as indicated at 37 °C for 10 min. The asterisk denotes a nonspecific signal from an antigen that was concentrated in this partially purified fraction. C, intensities of the p94 bands (B, closed arrowhead) were quantified, and the relative activities were plotted as a function of [NaCl] at a given [CaCl2]. The [NaCl] that gave half-maximal activity (Ka(Na+)) at [CaCl2] = 100 nm was ∼15 mm. D, skeletal muscle primary culture cells from WT (lanes 1–7) or p94CS-KI (8–10) mice were grown in differentiation medium for 6 days to form myotubes (
) and incubated without (lanes 1 and 8) or with 1 μm (lane 2), 5 μm (lane 3), or 10 μm (lanes 4 and 9) monensin or 10 μm (lane 5), 100 μm (lane 6), or 1 mm (lane 7 and 10) ouabain. Cells were harvested after 1 h. anti-pIS2 and CBB under the panels indicate detection was by Western blot analysis using anti-pIS2 and by Coomassie Brilliant Blue staining, respectively. Closed and open arrowheads indicate the full-length and autolytic fragments of p94, respectively, detected by an anti-pIS2 antibody.
FIGURE 4Western blot analysis of p94 mutants expressed in COS7 cells. Where indicated, the lysates of the cells expressing p94 mutants were incubated with 2 mm EDTA (E), 10 mm CaCl2 (Ca), or 150 mm NaCl (Na) at 37 °C. Vc indicates the negative control using an empty vector. Closed and open arrowheads indicate the full-length and autolytic fragments of the p94 mutants, respectively, detected by an anti-pIS2 or anti-p94-dI antibody, as indicated. Note that plural autolytic sites exist in the IS1 and IS2 of p94, and the Mr of the autolytic fragments varies depending on mutations (
). A, IS deletion mutants, Δex6 (= ΔIS1), Δex15, 16 (= ΔIS2), and Δex6, 15, 16 (= Δ(IS1+IS2)), were incubated in the absence (Substrate (C129S), −) or presence (Substrate (C129S), +) of p94:C129S as an intermolecular autolytic substrate. This is because IS1 deletion almost completely suppressed the intramolecular autolysis (lanes 7 and 8). Note that Δ(IS1+IS2) compromised the Na+, but not Ca2+, dependence of the mutant (lanes 27 and 28). Gray arrowheads indicate the full-length of the substrate p94:C129S (94 kDa). B, the spontaneous autolytic and fodrinolytic (lower panel) activities of p94 C-terminal deletion mutants, Δ(IS2+dIV) and Δ(dIII+IS2+dIV), were compared with those of p94 WT (lane 2) and their C129S double-mutant versions (lanes 4 and 6). Qualitative evaluations of autolysis and fodrinolysis are indicated by + and −. Although Δ(dIII+IS2+dIV) showed neither spontaneous autolysis nor fodrinolysis, it showed Ca2+- but not Na+-dependent autolysis (lanes 8 and 9), which was also blocked by EDTA (lane 7) as in the case of Δ(IS1+IS2) (A, lanes 25–28)) (
Kakinuma K.
Irie A.
Nakagawa K.
J. Biol. Chem. 2004; 279: 2761-2771
). C, involvement of CBS-IIa in activation of p94 by Na+ and Ca2+ was examined using a series of p94 mutants as in B. None of the single mutants in CBS-IIa or -IIb (lanes 3–6) showed substantially compromised activities of spontaneous autolysis and fodrinolysis, strongly suggesting that they retained both their Na+ and Ca2+ dependence, which could not be examined further by incubation due to the low signal level as in the case of WT. In contrast, the double mutant in CBS-IIa, D120A/E199A (lane 7), lost both the activities, but that in CBS-IIb (E364A/D371A, lane 8) did not. D, shown is Ca2+ and Na+ dependence of the autolysis in D120A/E199A and E364A/D371A compared with that of p94:C129S and p94:N358D. Again, D120A/E199A completely lost both its Ca2+ and Na+ dependence (lanes 13 and 4), but E364A/D371A retained them though very weak (lanes 17 and 18).
For intramolecular autolysis assay, they were incubated in the presence of 2 mm EDTA/KOH (pH 8.0), 10 mm CaCl2, or 150 mm NaCl at 37 °C for 40 min (Figs. 4, B, right panel, and D). To evaluate "intermolecular autolytic" activity of IS1 deletion mutants,110 of the amount of the lysate prepared from cells expressing p94:C129S mutant was added before the incubation (Fig. 4A).
Spontaneous autolysis of p94 and fodrinolysis by p94 were detected by Western analysis of the cell lysates without incubation using an anti-pIS2 antibody (for all except for C-terminal deletion mutants, which do not contain the antigen region), an anti-p94-dI antibody (Abcam, ab38960; for C-terminal deletion mutants), and an anti-Gly-Met-Met-Pro-Arg (GMMPR) antiserum, which was raised for this study using the penta-peptide (NH2-GMMPR-COOH) corresponding to the N-terminal sequence of calpain-proteolyzed fodrin as previously described (
Yokota M.
Nagao S.
Yamaura I.
Tani E.
Tsuchiya T.
Kawashima S.
) (Figs. 4, B, left panels, and C).
Construction of p94 Mutant Expression Vectors (Figs. 3B and 4)
IS1 and/or IS2 deletion mutants were produced using human WT p94 cDNA, as previously described (
). Missense mutants were produced as previously described (
). C-terminal deletion mutants were produced by introducing a termination codon into Ser-581 or Val-573 using the same mutation protocol as for the missense mutants. These cDNAs were inserted into a pSRD vector and expressed in COS7 cells as previously described (
FIGURE 3Regions responsible for the Na+ dependence of p94. A, alignment of the amino acid sequences of nine typical human calpains is shown. Parts of the protease subdomains IIa and IIb, which include the residues responsible for Ca2+ binding (indicated by #; named CBS-IIa and CBS-IIb, respectively) are shown. The residues shown in gray on a black background were mutated in subsequent experiments. Residues conserved among more than seven sequences are shown on a black background. Residue numbers for μCL/calpain1 are given above the sequences. Residue numbers for p94/calpain3 and calpainopathy-related mutations (from Leiden Muscular Dystrophy pages) are shown below the alignment (see also supplemental Fig. S1). B, summary of mutants used in this study and the dependence of their autolysis on Na+ and Ca2+ (see Fig. 4). For ΔIS mutants corresponding to splicing isoforms, Δex6, Δex15,16, and Δex6,15,16, the dependence was evaluated by their proteolytic activity against p94:C129S (*, see Fig. 4A). The dependence of the Δ(IS2+dIV) mutant was determined by comparing its level of expression and fodrinolytic activity with those of the Δ(IS2+dIV)/C129S double mutant (**, see Fig. 4B). Note that ΔIS2, but not Δ(IS2+dIV), can be detected by anti-pIS2 as ΔIS2 retains a part of the antigen region for anti-pIS2.
Muscle Cell Cultures
The preparation of mouse skeletal muscle primary cell cultures, and their transfection were carried out as previously described (
). They were incubated with or without the indicated concentrations of monensin or ouabain. The cells were harvested after 1 h and subjected to Western blot analysis using the anti-pIS2 antibody (Fig. 2D). For real-time observation of the p94 autolysis (supplemental Videos S1 and S2), the full-length human WT p94 cDNA and its protease-inactive form, p94-C129S, were cloned into the pEGFP vector (Clontech). Four days after switching to differentiation medium, myocytes were subjected to analysis. To increase the [Na+]i, 1 mm ouabain was added to the culture medium. Images were scanned and recorded using an LSM510 confocal microscope (Zeiss) equipped with an incubation system (37 °C, 5% CO2; Tohkaihit Co.).
Proteomic Analysis (FIGURE 5, FIGURE 6, FIGURE 7)
Skeletal muscle (GC) extracts from WT and p94CS-KI mice (two mice for each species) were prepared by homogenizing them in 20 mm triethylammonium bicarbonate, 1 mm EDTA/K+, 1 mm Tris-(2-carboxylethyl) phosphine, and 1 mm phenylmethylsulfonyl fluoride, as described previously (
). The protein concentrations were determined for each sample, and equal amounts of two WT samples were mixed and used for further analysis. The p94CS-KI samples were treated similarly, so that both the WT and p94CS-KI samples had the same protein concentration (final 7.5 mg/ml). They were then incubated at 37 °C for 10 min with 150 mm NaCl or CsCl with 1 mm EDTA/KOH (pH 8.0) or 2 mm CaCl2 or MgCl2 with 0.15 mg/ml calpastatin (to suppress the activities of the conventional calpains; Takara). After the incubation, the Sup and Ppt fractions were obtained by centrifugation (20,000 × g, 30 min, 4 °C). Each fraction was then completely digested by trypsin (37 °C, 15 h) followed by labeling with iTRAQTM 8-plex reagent (ABI) according to the manufacturer's instructions. The samples were then analyzed using two-dimensional-liquid chromatography tandem mass spectometry as previously described (
Shindo M.
Kudo K.
Tsubata T.
Biotechnol. J. 2007; 2: 565-576
). Peptides and proteins were identified by ProteinPilotTM Version 3 using a confidence value of 0.7 (80%). Ratios were calculated from quantified iTRAQTM signals: RWT-Na = iTRAQTM113/iTRAQTM118, RCS-Na = 119/121, RWT-Ca = 114/115, RCS-Ca = 116/117, RNa = RWT-Na/RCS-Na = 113/118/(119/121), RCa = RWT-Ca/RCS-Ca = 114/115/(116/117), RCs = 118/121. Candidates for substrates were selected as those showing a significant decrease (RWT-Na < 0.8, RWT-Ca < 0.8) after incubation with NaCl or CaCl2 compared with the negative control (CsCl or MgCl2, respectively) only for WT but not for p94CS-KI (RCS-Na > 0.8, RCS-Ca > 0.8, or RWT-Na/RCS-Na < 0.7, RWT-Ca/RCS-Ca < 0.7). (see Fig. 5A). The activation and autolysis of p94 was confirmed by Western analysis of each fraction (before separation by centrifugation) using the anti-pIS2 antibody and Super Signal West Dura detection (Thermo) (Fig. 5B). The rationale for selecting candidates for in vitro and in vivo substrates is shown schematically in Fig. 5C.
FIGURE 5Scheme for the differential proteome analysis to determine p94 substrates specific for the activating ions. A, total extracts from the GC muscles of WT and p94CS-KI mice were incubated in the presence of NaCl or CsCl and EDTA or of CaCl2 or MgCl2 and calpastatin. The samples were spun to obtain the Sup and Ppt fractions, digested by trypsin, and labeled with iTRAQTM 8-plex reagent. The labeled samples were mixed and analyzed by two-dimensional/tandem mass spectometry, and peptides/proteins were identified by ProteinPilotTM. Candidates for substrates were selected as those showing a significant decrease after incubation with NaCl or CaCl2 compared with the negative control (CsCl or MgCl2, respectively) only for WT but not for p94CS-KI, as indicated in the equations. B, shown is a Western blot analysis of the above samples using anti-pIS2. The above samples (total extracts before centrifugation) before (lanes 1–4) or after (lanes 5–12) incubation with CsCl (lanes 5 and 7), NaCl (lanes 6 and 8), MgCl2 (lanes 9 and 11), or CaCl2 (lanes 10 and 12) were examined by Western blot analysis using the anti-IS2 antibody. The upper and middle panels show 30-s and 3-min exposures. Asterisks denote nonspecific signals. The lowest panel, the 210-kDa myosin heavy chain stained by Coomassie Brilliant Blue (BBC), shows that each sample had a different protein concentration before the incubation (the second WT sample was especially skewed). The amounts of p94 in the WT and p94CS-KI mouse muscle were roughly equal (see Fig. 2A). C, theoretical signal patterns for p94 substrates that were recognized in a Na+-specific (1), Ca2+-specific (2), or Na+/Ca2+ (3) manner (for a list of substrates, see Fig. 6 as indicated). If substrates were constitutively proteolyzed in vivo, their amounts should be lower in WT than in p94CS-KI muscle without in vitro p94 activation. Therefore, they should be lower in WT-CsCl than in p94CS-KI-CsCl (4∼6). For candidate in vivo substrates, those identified by only a single kind of peptide were eliminated to avoid false positives (for a list, see Fig. 7). The numbers in the right-hand column indicate the number of proteins in each fraction (Sup or Ppt) that showed the corresponding patterns.
FIGURE 6Summary of proteins that showed a Na+- and/or Ca2+-dependent decrease in WT, but not p94CS-KI, mouse skeletal-muscle fractions. A, Na+-dependent substrate candidates corresponding to those shown in Fig. 5C, 1 or 3. The lists were sorted by RWT-Na (the ratio of protein in NaCl to that in CsCl for WT mice corresponding to iTRAQTM signals at 113 and 118 for WT (see Fig. 5A). WT/CS is the ratio of the values obtained for WT and CS by each normalization (RWT-Na/RCS-Na and RWT-Ca/RCS-Ca, respectively). B, Ca2+-dependent substrate candidates corresponding to those shown in Fig. 5C, 2 or 3. Lists were sorted by RWT-Ca. Red, orange, sky blue, or dark blue backgrounds indicate that the values were less than 1/1.5 (0.67) or 1/1.25 (0.8) or more than 1.25 or 1.5, respectively. No. pep. indicates the number of kinds of peptides used to identify the corresponding protein.
FIGURE 7Potential in vivo substrates for p94 differentially activated by Na+ and Ca2+. Candidate in vivo substrates are selected from Fig. 6. The rationale for selecting candidates is shown schematically in Fig. 5C, 4–6. Black and gray backgrounds indicate values <1/1.5 (0.67) and <1/1.25 (0.8), respectively. No. pep. indicates the number of kinds of peptides used to identify the corresponding protein.
An Activity-attenuated Mutant of Skeletal Muscle-specific Calpain, p94/Calpain 3, Showed Na+-dependent Autolysis
In contrast to conventional calpains, whose autolysis is suppressed by high salt concentrations (
Biochim. Biophys. Acta. 2004; 1691: 91-103
), autolysis of p94:N358D was NaCl-dependent (Fig. 1A). Surprisingly, KCl did not cause significant autolysis (Fig. 1A), indicating that the p94 autolysis was specifically dependent on Na+ and was not a general response to high salt concentrations. This explains the previous in vitro translation results showing that WT p94 undergoes autolysis in the absence of Ca2+ and the presence of NaCl (
). As described above, p94:N358D also shows Ca2+-dependent autolysis (Fig. 1B) (
). Ten millimolar CaCl2 and 150 mm NaCl were similarly effective (Fig. 1C). Notably, the autolysis patterns of p94 were different when induced by Ca2+ or Na+, suggesting that p94:N358D assumed distinct conformations when activated by the two ions. The [Na+] and [Ca2+] required for autolytic activity were ∼100 mm and 100 μm, respectively, in the absence of other ions (Fig. 1, A and B).
Native WT p94 Also Showed Na+-dependent Autolysis
We next examined whether native WT p94 showed the same activity profile. As a control, we used p94:C129S "knock-in" (p94CS-KI) mice, which have a missense mutation corresponding to C129S in Capn3 exon 3 and express a protease activity-null p94 mutant p94:C129S in place of WT p94.6 Total lysates of the skeletal muscles from WT and p94CS-KI mice were prepared in the absence of Ca2+ and Na+ and separated to Sup and Ppt fractions by centrifugation. Comparable levels of the 94-kDa pre-autolytic band were detected in both preparations and both fractions (Fig. 2A, lanes 1, 2, 17, and 18). When incubated with Ca2+ or Na+, the p94 expressed in WT muscle, but not the p94:C129S expressed in p94CS-KI muscle, autolyzed (lanes 3–8 and 19–24). When the incubations were carried out with both ions at varying concentrations using WT p94 partially purified from rat GC (supplemental Fig. S2), the ion effects were additive; that is, the [Na+] required for half-maximal autolytic activity was 15 mm at [Ca2+] = 100 nm, indicating that physiological cytosolic concentrations of Na+ and Ca2+ can activate WT p94 (Fig. 2, B and C).
As a more physiological condition, primary cultures of skeletal muscle cells from WT and p94CS-KI mice were treated with monensin or ouabain. Monensin is an ionophore specific for monovalent metal ions (Na+ ≫ K+ > Rb+) (
Yu X.M.
Salter M.W.
), and ouabain is a specific inhibitor of the Na+/K+-ATPase (
). Thus, both drugs increase [Na+]i, but by different mechanisms. When myocytes were incubated with either drug, p94 was autolyzed in the WT cells but not in the p94CS-KI cells (Fig. 2D). This indicated that p94 is activated by a small increase in [Na+]i under nearly physiological conditions. The increase in [Na+]i also caused increased [Ca2+]i, and it is also possible that the combined increase of [Na+]i and [Ca2+]i caused the autolysis of WT p94.
To investigate this phenomenon in real time, green fluorescent protein-fused WT p94 or p94:C129S was expressed in differentiated myocytes (myotubes) from WT or p94CS-KI mice, respectively, and time-lapse imaging was performed. The exogenous green fluorescent protein-p94s (both WT and p94:C129S) were predominantly observed at the M-line (
). When ouabain was added to the medium, the green fluorescent protein signals of p94WT, but not those of p94:C129S, disappeared within 30 min, indicating the rapid autolysis of p94 in the cell was stimulated by the increase in [Na+]i (see the supplemental videos and Fig. S5, A and B).
One of the Ca2+-binding Sites in the p94 Protease Domain Is Required for Na+-binding
To determine the region of p94 responsible for its Na+-dependent activation, we constructed various mutants (Fig. 3) and examined their Na+-dependent activities. The domain structure of p94 is typical of the conventional calpains (domains I to IV; dI∼dIV, see Fig. 3B and supplemental Fig. S1), and the sequences of its Ca2+-binding sites are well conserved (Fig. 3A). In addition, alternatively spliced exon 6 and exons 15–16, respectively, encode two insertion sequences in p94, IS1 and IS2, that are responsible for rapid autolysis of p94 (
Kimura E.
Montarras D.
Pinset C.
Mol. Cell Biol. 1999; 19: 4047-4055
We first examined the autolysis of a mutant p94 lacking the IS1 and/or IS2 regions. Because the deletion of IS1 eliminates the major autolytic sites, p94:C129S was used as an alternative intermolecular autolytic substrate for the assay. In cultured cells, not only p94 itself, but also co-expressed p94:C129S is "intermolecularly autolyzed" by WT p94 (
Kinbara K.
Kimura S.
Takahashi M.
Sasagawa N.
Sorimachi N.
Tagawa K.
Maruyama K.
). ΔIS2 but not ΔIS1 showed a moderate reduction in Na+ dependence, and the Na+-dependent autolysis of Δ(IS1+IS2) was almost completely suppressed (Fig. 4A, lanes 12, 20, and 28). On the other hand, the expression of a C-terminal deletion mutant, Δ(IS2+dIV), showed spontaneous autolysis and fodrinolysis, similar to WT (Fig. 4B, lanes 2 and 3), i.e. at physiological [Na+]i + [Ca2+]i. Fodrin is one of in vivo substrates of conventional calpains and is also a good substrate for p94, and thus, the endogenous fodrin is proteolyzed at a specific position when p94 is expressed in cultured cells (
). However, Δ(dIII+IS2+dIV), which is 8 amino acids shorter than Δ(IS2+dIV), lost both its autolytic and fodrinolytic functions (Fig. 4B, lanes 5 and 6). Incubation of Δ(dIII+IS2+dIV) with Ca2+, but not Na+, caused autolysis (Fig. 4B, lanes 7–9). These results indicated that dIV is dispensable for the Na+ binding of p94, and IS1, IS2, and dIII are involved in Na+ binding, with IS2 playing the most essential role.
Next, we replaced several well conserved Ca2+ binding residues with Ala (Fig. 3). Surprisingly, none of the single mutations (D120A or E199A, which correspond to the Ca2+-binding site in dIIa (CBS-IIa) or E364A or D371A in dIIb (CBS-IIb)) substantially compromised the spontaneous autolysis or fodrinolysis of p94 (Fig. 4C and supplemental Fig. S3). However, the double mutation D120A/E199A (in CBS-IIa) was insensitive to both Na+ and Ca2+, and E364A/D371A (in CBS-IIb) showed weak Na+- and Ca2+-dependent activity (Fig. 4D and supplemental S3). These results (summarized in Fig. 3B) strongly suggest that CBS-IIa is important for the p94 activity and that Na+ and Ca2+ both act at CBS-IIa.
We also tested 37 missense p94 mutants (including the ones described above), 3 of which corresponded to calpainopathy causative mutations. None showed Na+-dependent, Ca2+-insensitive autolysis (supplemental Fig. S4). This strongly suggests that the effect of Na+ is transduced via one of the Ca2+-binding sites. Altogether, our data indicated that IS1, IS2, and dIII contribute to the CBS-IIa-mediated Na+-dependence of p94 (supplemental Fig. S3). The involvement of other Ca2+-binding sites, however, cannot be completely eliminated. In any case, our findings suggest that the Na+ dependence of p94 is achieved by the insertion of IS1 and IS2 in the p94 molecule, which may change the relative conformations of dII, dIII, and dIV.
Na+ and Ca2+ Give p94 Differential Substrate Specificities, at Least in Vitro
Finally, to shed light on the physiological meaning of Na+ dependence of p94, we screened for p94 substrates by differential proteomic analysis using iTRAQTM. Extracts from WT and p94CS-KI skeletal muscles were incubated with NaCl, CsCl, CaCl2, or MgCl2, and 996 proteins were identified and quantified (Fig. 5A). Under the conditions used, Na+ and Ca2+ caused complete p94 autolysis, whereas Cs+ and Mg2+ resulted in no and weak autolysis, respectively (Fig. 5B). Among the proteins identified, candidates for substrates were selected as those showing a significant decrease (<×0.8) after incubation with NaCl or CaCl2 compared with their negative control (CsCl or MgCl2, respectively) in WT extract but not in p94CS-KI extract (Fig. 5C, 1–3). Relatively few (11∼29) proteins were identified for each ion and fraction. Strikingly, there was almost no overlap between the candidate substrates identified by incubation with Na+ versus Ca2+ (Fig. 6, A and B). Although these proteins may not have in vivo relevance, our data show that p94 has different substrate specificities in vitro depending on whether it is activated by Na+ or Ca2+ and on how these ions affect the substrates themselves.
Possible in Vivo Substrates for p94 Activated by Na+ and/or Ca2+
It is reasonable to think that the normal in vivo substrates for p94, if any, might be less abundant in muscle cells expressing WT p94 than in those expressing p94:C129S. To look for candidate in vivo substrates, we compared the levels of proteins present in the negative controls (WT/CS in CsCl incubation) to find those that were significantly reduced in muscle from WT mice compared with p94CS-KI mice (WT/CS-CsCl < 0.8 and NaCl/CsCl-WT < 0.8 for candidates for Na+; WT/CS-CsCl < 0.8 and CaCl2/MgCl2-WT < 0.8 for candidates for Ca2+, Fig. 5C, 4–6). This analysis yielded three candidate in vivo substrates for Na+-activated p94, three for Ca2+-activated p94, and one for p94 activated by either ion (Fig. 7). Notably, these candidates included several myofibrillar proteins, such as tropomyosin, troponins T and I, and α-actinin-3, suggesting roles for p94 in modulating the amounts of these proteins and, hence, in myofibril quality control. It is also noteworthy that calsequestrin-1, a Ca2+-binding/storing protein in the SR, is a candidate substrate for p94 activated by either ion. That calsequestrin probably has different conformations in the absence and presence of Ca2+ suggests p94 recognizes different substrates depending on whether it is activated by Na+ or Ca2+. On the other hand, it is possible that tropomyosin and troponins, also Ca2+-binding proteins, show different proteolytic patterns because they change their conformation depending on the presence or absence of Ca2+ rather than on a difference in substrate recognition of p94.
Because conventional μ- and m-calpains require ∼50 μm and 1 mm Ca2+, respectively, for their activity (
), they can rarely be fully active in the cytosol, which may be a safety mechanism to prevent their overactivation. Although it is unclear which is the cause and which the result, the physiological [Na+]i-dependent low Ca2+ requirement of p94 is a property that facilitates its responsiveness to physiological [Ca2+]i changes in skeletal muscle. The rapid autolytic degradation of p94 (
Guyon J.R.
Potts A.
Chamberlain J.
Dalkilic I.
Kunkel L.M.
), on the other hand, probably serves as a blockade against its overactivation. It is also possible that, at other times, a suppressor(s) of p94, which includes the connectin/titin N2A region (
Huebsch K.A.
Wooley C.M.
Sher R.B.
Seburn K.L.
Cox G.A.
), attenuates p94 activity, enabling it to function immediately by changing its conformation in response to alterations in [Na+]i and/or [Ca2+]i without autolysis or the proteolysis of substrates.
In this respect, it is noteworthy that p94 is present in the SR (
Wu B.
Ottenheijm C.
Granzier H.
). Although not generally high in the cell, the [Na+]i increases transiently immediately beneath the sarcolemma, during the depolarizing end-plate potential caused by the neuromuscular transmitter acetylcholine. This depolarization leads to increased [Ca2+]i as Ca2+ is released from the SR into the cytosol, but the excitation-coupled contraction is not accompanied by the influx of Ca2+ from outside the cells in most skeletal muscles (
Flucher B.E.
Franzini-Armstrong C.
). Instead, the sarcolemmal depolarization causes conformational changes in the skeletal muscle-specific dihydropyridine receptor at the T-tubule membrane, which interacts directly with the ryanodine receptor (RyR) at the SR membrane to elicit Ca2+ efflux from the SR (
Tanabe T.
Beam K.G.
Adams B.A.
Niidome T.
Numa S.
Cheng W.
Altafaj X.
Ronjat M.
Coronado R.
). However, it is largely unknown how depolarization causes the dihydropyridine receptor to change its conformation, and it is tempting to speculate that p94 at the SR membrane could contribute by sensing fluctuations in the local [Na+]i and/or [Ca2+]i. Indeed, p94 is predominantly expressed in skeletal, and not heart, muscle (
), and p94-KO mice have an impaired Ca2+ efflux from intracellular stores, but the influx of extracellular Ca2+ is not impaired (
Several proteases involved in blood coagulation have Na+-dependent activity (
). In particular, factor Xa (FXa) has one Ca2+- and one Na+-binding site (see Fig. 8). These sites are allosterically linked and have additive effects, although the concentrations required for activity of FXa are much higher than that of p94 because FXa is equipped to respond to extracellular conditions (apparent dissociation constant for [Na+] of FXa is 116 mm when [Ca2+] is 2.5 mm) (
Rezaie A.R.
He X.
Biochemistry. 2000; 39: 1817-1825
). FXa is a serine protease whose primary structure has no similarity to that of the calpain protease domain (
Hanna R.A.
Campbell R.L.
Davies P.L.
). Nevertheless, the loops responsible for the Ca2+/Na+ binding are similarly positioned relative to the active-site cleft in these two proteins (Fig. 8), which may be informative about their evolution.
FIGURE 8Three-dimensional structures of factor Xa and the μ-calpain/calpain 1 protease domain. Cross-eye stereo views were drawn by FiatLux MolFeat Version 4.5 using Protein Data Bank codes 2VVU for factor Xa (A) and 1KXR for Ca2+-bound μ-calpain (B). The active-site residues are indicated by the ball-and-stick drawings in the orange circles. The blue and yellow balls represent Ca2+ and Na+. The Ca2+ binding residues of the μ-calpain protease domain and those corresponding to the ones used for the mutagenesis study (see Fig. 3) are indicated by blue and pink ball-and-stick drawings, respectively. The position of the IS1 insertion in p94 is indicated by the blue arrow.
Because the metal ion radii of Ca2+ and Na+ are almost identical, a slight modification might enable a Ca2+-binding site to bind Na+. Indeed, Bonagura et al. (
Bonagura C.A.
Bhaskar B.
Sundaramoorthy M.
Poulos T.L.
Pappa H.S.
Patterson W.R.
) reported that artificial mutagenesis of plural amino acid residues of cytochrome c peroxidase (CCP) makes CCP K+-, Na+-, or Ca2+-dependent. Our mutagenesis study (see Fig. 3) also strongly suggested that p94 CBS-IIa acquired its Na+-sensing ability by undergoing conformational changes induced not only by IS1 and residues in the vicinity of CBS-IIa but also by IS2 and dIII. This suggests that a large portion of the p94 molecule is involved in making p94 Na+-dependent. Therefore, various mutations along the entire p94 molecule can potentially destroy proper activity of p94, leading to calpainopathy. This may explain why calpainopathy patients have missense mutations scattered along the entire p94 molecule rather than clustered in the protease domain (
) (see supplemental Fig. S1).
The evolutional process that led to acquisition of Na+ binding ability of p94 is unknown but interesting to contemplate. Obviously, the insertion of IS1 and IS2 by the addition of exons 6, 15, and 16 represents one or more important events. All vertebrates, but no other living organisms so far examined, have highly conserved CAPN3 genes (supplemental Fig. S1 and Table S1). The deduced amino acid sequence identity between the p94 paralogues of the most distant vertebrates, humans, and puffer fish, is 61.6% (supplemental Table S2), which is comparable with the interspecies identities of the representative intracellular proteases cathepsin L (56.4%), caspase 3 (64.4%), and proteasome subunit β5 (71.0%) (
Mizushima N.
Autophagy. 2007; 3: 179-180
). This level of sequence identity implies that p94 is indispensable in the skeletal muscles of all vertebrates. Strikingly, the N-terminal half of the IS2 region, one of the characteristic p94 protein domains, is highly conserved in all vertebrates (supplemental Fig. S1), and three human calpainopathy-causative mutations (S606L, R608K, and A609E) are found as a cluster in this region.
The protease domain (subdomains IIa+IIb) and domain III are also highly conserved across vertebrate species, but subdomain IIb is slightly less conserved than the two others. In particular, IIa and IIb differ most in their Ca2+-binding sites; i.e. five of eight residues constituting CBS-IIa are conserved in all sequences, whereas two of seven are conserved in CBS-IIb (supplemental Fig. S1). The importance of CBS-IIa is also supported by the fact that Thr-192 and Lys-193 adjacent to CBS-IIa are conserved in all p94s but not in any other vertebrate calpain (supplemental Fig. S1) (
J. Biochem. 2001; 129: 653-664
). Accordingly, calpainopathy-associated mutations have been identified in four of the CBS-IIa residues and Thr-192 (D120N, S194C, E199Q, W201R, and T192I), but none has been found in the CBS-IIb residues (supplemental Fig. S1).
Altogether, these findings support the importance of IS2, CBS-IIa, and domain III for p94 function. On the other hand, the IS1 sequences are rather divergent. Considering our finding that p94:ΔIS1 had almost no effect on the Na+/Ca2+ dependence (see Figs. 3B and 4A) except for a loss of intramolecular autolysis, IS1 probably provides the intramolecular proteolytic sites that allow rapid autodegradation. In this sense, the amino acid sequence itself may not be so important, and any unstructured oligopeptide may function as the IS1. Then, why did vertebrates need an additional unique calpain, p94? Generally, vertebrates are terrestrial and larger than invertebrates, and thus, their skeletal muscles carry much higher loads/stress. It is possible that p94 is involved in the stress response of vertebrate skeletal muscles, e.g. by regulating stress-responsive proteins such as heat-shock proteins.
In light of our results, we propose three functional roles for Na+ dependence of p94; 1) the Na+ dependence could make p94 more responsive than conventional calpains to physiological changes in [Ca2+], 2) substrate specificity of p94 could be finely regulated by the balance between [Na+] and [Ca2+], and 3) a conformational change in p94 upon its binding to Na+/Ca2+ could be propagated through its interaction with structural components. Our findings are important for understanding how pathogenic/dystrophic situations are normally circumvented in skeletal muscle, and they may contribute to the development of novel therapeutic and diagnostic strategies for muscular dystrophies. At a minimum, our finding also provides the useful information that patient muscle samples should be analyzed in Na+-free buffers when a diagnosis of calpainopathy is suspected. Otherwise, p94 is rapidly degraded, which can result in a misdiagnosis. On the other hand, comparison of incubations of skeletal muscle extracts from patients in buffers with and without NaCl will provide us with simple and accurate detection of p94 activity, i.e. diagnosis of calpainopathy, which has been already suggested previously (
Fanin M.
Nascimbeni A.C.
Angelini C.
J. Med. Genet. 2007; 44: 38-43
). Our results also reveal a new essential function of Na+ in the cytosol and raise the possibility that Na+ is involved in regulating other cytosolic Ca2+-dependent enzymes.
We thank all the members of the Calpain Project laboratory (Rinshoken) and Laboratory of Biological Function (University of Tokyo) for valuable support and discussions.
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Received in revised form: May 3, 2010
We are most grateful to Dr. Koichi Suzuki (Professor Emeritus of The University of Tokyo), who established the basis of our calpain studies including this study and passed away on April 20, 2010. We dedicate this paper to the memory of him and will never forget his mentorship and generosity.
Author's Choice—Final version full access.
DOI: https://doi.org/10.1074/jbc.M110.126946
© 2010 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
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Creative Commons Attribution (CC BY 4.0)


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Home/NEWS/China fires a salvo of criticism at US on HK, protests, wanting to quit WHO
China fires a salvo of criticism at US on HK, protests, wanting to quit WHO
Socially Keeda Send an email June 1, 2020
China on Monday fired a salvo of criticism against the US, threatening Washington with "firm countermeasures" if it harms Chinese interests in Hong Kong and saying America is "addicted to quitting" because of President Donald Trump's decision to leave the World Health Organisation (WHO).
Chinese foreign ministry spokesperson, Zhao Lijian, referring to the largescale protests and riots following the death of African-American George Floyd, added that racial discrimination is a long-standing problem in the US and Beijing hoped the US government would take all necessary measures to deal with the violent law enforcement of police, so as to protect and safeguard the legitimate interests of racial minorities.
Black lives matter and their human rights should be protected, he said.
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Current ties between the two countries have increasingly soured in recent months over several issues including on the origins of the coronavirus, arms sales Taiwan and ongoing disputes over trade.
On Hong Kong, Zhao said any move by the US to harm Chinese interests will be met with firm countermeasures, questioning Washington's decision to begin ending special treatment for Hong Kong as well as actions against Chinese students and companies.
Trump ordered his administration to begin the process of eliminating special US treatment for Hong Kong in response to China's parliament last week voting to implement a new national security legislation on Hong Kong, which, critics say, could impact the "one country, two systems" mechanism.
The US President, however, hasn't mentioned a timeline for the withdrawal of the special status.
"The announced measures severely interfere with China's internal affairs, damage U.S.-China relations, and will harm both sides. China is firmly opposed to this," Zhao said at the regular ministry briefing on Monday.
"Any words or actions by the U.S. that harm China's interests will meet with China's firm counterattack," he said.
Earlier, Hong Kong's Beijing-backed government was quoted in news reports as telling the US to keep out of the national security debate, warning that withdrawal of the financial hub's special status could backfire on the US economy.
On Trump's announcement that the US would leave the WHO, Zhao said the "…US has become addicted to quitting groups and scrapping treaties".
Trump announced on Friday the US would cut ties with the WHO, accusing the UN agency of becoming China's puppet.
America china China US China US ties criticism fires Hong Kong Jaipur news latest news jaipur President Donald Trump protests quit salvo socially keeda news United States of America US wanting WHO World Health Organisation Zhao Lijian
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redpajama_set_name: RedPajamaCommonCrawl


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Dental Excellence of Napoleon: Dentistry From the Heart–Spreading Free Smiles!
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Are you or someone you know struggling to afford dental care? Join us for Dentistry From the Heart coming up Friday, May 10th!
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text:
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{
    public class AccountOpeningBalance
    {
        public int AccountId { get; set; }
        public int ID { get; set; }
        public decimal Balance { get; set; }
        public string Reason { get; set; }
        public DateTime Date { get; set; }
    }
}

redpajama_set_name: RedPajamaGithub


TCM has announced it's full line up for it's TCM Road To Hollywood tour. I love that they make this effort to bring classic film and it's stars to people around the country, as a build up to the amazing TCM film festival in Hollywood.
And just to keep us guessing they still have one date, city and screening to be determined.
Yep! I'll be going to see RIO BRAVO with 650 of my closest friends at the Historic Texas Theatre in Dallas. Yes, THAT Texas Theatre - the one where Lee Harvey Oswald was watching WAR IS HELL when they arrested him in 1963.
So it will be interesting to see Angie Dickinson there with Ben Mankiewicz that night. We can't wait!!
I'll look forward to hearing about it when I see you at the film fest.


text:
The original story can be found at http://www.bpnews.net/30503/nh-gov-reneges-on-gay-marriage
NH gov. 'reneges' on 'gay marriage'
by Michael Foust, posted Friday, May 15, 2009 (10 years ago)
CONCORD, N.H. (BP)--Disappointing conservatives who say he is reneging on a previous statement to voters, New Hampshire Democratic Gov. John Lynch said Thursday he would sign a bill legalizing "gay marriage" if changes to it are made to protect religious convictions.
Lynch, who as recently as April said he believed marriage was "reserved" for one man and one woman, released a statement saying he wants to see the bill's protections for religious organizations strengthened. Opponents, though, said the governor's proposal falls far short and that even if he gets his way, parental rights and religious freedoms still will be impaired, with "gay marriage" being taught as normative in public schools.
Lynch said he would veto the bill, H.B. 436 in its current form, although it has yet officially to make it to his desk and it appears legislators simply will hold off on sending it there until it is tweaked to incorporate Lynch's proposals. It already has passed the state House and Senate. A Senate leader told the Union-Leader newspaper that the changes could be made as early as next week.
Two polls in the past week -- one by a non-partisan polling agency and one released by an opponent of the bill -- showed at least a plurality of opposition to "gay marriage" in the state.
"Bottom line: Governor Lynch has broken his trust with the New Hampshire public by stating he will sign this bill, and has given in to the radical special interests," the conservative Cornerstone Policy Research said in an e-mail to constituents.
Ever since the bill passed the state senate May 6, supporters and opponents have flooded Lynch's office with phone calls, something Lynch acknowledged in his statement. Cornerstone Policy teamed up with the National Organization for Marriage and ran TV ads showing Lynch's past statements opposing "gay marriage" and urging him to stick to his word.
Opponents now will turn their attention back to the legislature and pressure senators and representatives to oppose the bill. It squeaked through both chambers, passing the Senate in an initial vote 13-11 and the House 186-179, also in an early vote. In fact, the bill actually lost by one vote in its very first test in the House, although it was revived minutes later when several representatives changed their positions.
"My personal views on the subject of marriage have been shaped by my own experience, tradition and upbringing," Lynch said. "But as Governor of New Hampshire, I recognize that I have a responsibility to consider this issue through a broader lens."
Lynch said he had met with legislative leaders and given them proposed bill language that he said would "provide the strongest and clearest protections for religious institutions and associations, and for the individuals working with such institutions."
"It will make clear that they cannot be forced to act in ways that violate their deeply held religious principles," he said.
Lynch's proposed change would add language stating, in part, that "a religious organization, association or society" or any individual "who is managed, directed or supervised by or in conjunction with a religious organization, association or society" cannot be required to facilitate a "gay marriage" in any way.
Although that could protect, for instance, Christian schools, it falls far short of what really is needed, Cornerstone Policy Research said.
"This new language adds protections for religious organizations that are already covered under the First Amendment --- but still does nothing to protect independent business owners and persons of faith who are Justices of the Peace, who will now be forced to provide their services for gay 'marriage' ceremonies or risk being fined up to $50,000 by the Human Rights Commission," the group said.
Lynch's proposed changes also would not protect parental rights in public schools, critics say. New Hampshire's neighbor to the south, Massachusetts -- which also recognizes "gay marriage" -- has witnessed such school-related conflicts. In one instance, a school system told the husband and wife of a second-grader in Lexington, Mass., that the parents had no right to object to the reading of "King & King," a book about "gay marriage" in the child's class. The parents sued but lost in federal court.
The Becket Fund for Religious Liberty, which takes no position on "gay marriage," applauded Lynch for taking the step toward protecting religious freedom but said the proposed additions won't cover everything needed.
"Governor Lynch's conscience protections go significantly further than those set forth in states such as Vermont and Connecticut [two states where gay marriage is legal," the Becket Fund said in a statement. "Lynch's proposal would in some instances also protect individuals as well as institutions, though it would not protect small business owners such as the [New Mexico] photographer who faces a fine for refusing, in accordance to her religious beliefs, to photograph a same sex union."
Eric Rassbach, national litigation director of the Becket Fund for Religious Liberty, said, "Giving legal recognition to same-sex marriages promises to unleash a host of legal and financial penalties on those who conscientiously object to it, unless states make the effort to enact robust legal protections."
As recently as April, Lynch told The Union-Leader, "I think the word 'marriage' is reserved for a marriage between a man and a woman, and I think the real issues really are rights and protections for gay and lesbian couples."
A Dartmouth College poll released earlier this month showed 45 percent of registered voters opposing "gay marriage" and 41 percent supporting it. Cornerstone Policy Research also released a poll of 50,000 households showing that 64 percent agreed that "marriage between one man and one woman should be the only legal definition of marriage" in the state. A Virginia company called ccAdvertising conducted the survey.
The Union-Leader editorial board criticized Lynch, saying the governor had "handed unequivocal victory to proponents of same-sex marriage."
"[I]t opens the definition of marriage to perpetual revision henceforth," the editorial read. "Should the governor's proposal pass, marriage shall instantly transform from a bedrock of civilization to just another in our ever-expanding list of political battlefields. Its meaning is to be fought over, revised, changed, updated and expanded whenever the preferences of the majority shift direction. The specific ramifications for society are unpredictable, but the general shift is not. It will be a shift toward a legal structure in which anything goes. The damage will be widespread and irreversible. And Gov. Lynch, who reassured us all that on his watch 'marriage is going to continue as it has,' pulled the pebble that caused the avalanche."
New Hampshire would become the sixth state to legalize "gay marriage," although in two of those states the laws have yet to take effect. Vermont's law goes into effect in September and Maine's law likely won't go into effect until voters have a say. Just as significantly, if New Hampshire does change its laws, Rhode Island would be the lone state in New England not to redefine marriage.
Michael Foust is an assistant editor of Baptist Press. To read how "gay marriage" impacts parental rights and religious freedom visit http://www.bpnews.net/bpnews.asp?id=30209.


Spanish Studies with Chinese is taught by the Department of Languages and Cultures. Our distinctive approach enables you to acquire high-level language skills and a thorough understanding of Spanish and Chinese cultures and societies within a global context. Iberian Languages at Lancaster is ranked 5th in the UK by the Complete University Guide 2017.
Your first year comprises an exploration of the Spanish language and its cultural context. Alongside this, you will study an intensive Chinese language and culture module, designed for beginners with little or no prior knowledge. You will also take a minor subject of your choice, and this could be a third language.
In Year 2, you will study Spain's culture, politics and history in more depth, as well as selecting courses which are international in scope and promote a comparative understanding of Europe and beyond. In the core Chinese modules, you will be introduced to more complex grammar, key sentence patterns and expand your vocabulary.
Spending your third year abroad in a Spanish-speaking country makes a major contribution to your command of the language, while deepening your intercultural sensitivity. You can study at a partner institution or conduct a work placement. We strongly recommend spending time in China too as part of your degree and will support you in securing funding to do so.
In your fourth year, you will continue developing your Spanish and Chinese language skills alongside a course in Chinese culture and society. You will combine this with a choice of specialist culture and comparative courses. You will also develop a particular topic from your second year studies into your specialist dissertation.
Required Subjects A level Spanish and/or Chinese. Native Spanish or Mandarin speakers will not be accepted onto this scheme.
Following on from Part I Chinese, this module introduces you to more complex Chinese grammar and key sentence patterns. You'll increase your vocabulary range to help you interact more comfortably in more demanding situations.
This is achieved through four contact hours a week. You'll attend a two-hour seminar to practise listening and speaking. A one-hour lecture each week will teach you grammar and language functions. Reading practice, grammar exercises and character learning is covered in a weekly one-hour seminar.
You'll reach a standard beyond A-Level of the CEFR (Common European Framework of Reference for Languages).
This module progresses from Part 1 Chinese. It will introduce you to more complex Chinese grammar and key sentence patterns. You'll be encouraged to expand your knowledge of vocabulary to help you communicate in demanding interactive situations.
You'll develop a more independent approach to language learning and academic enquiry. There will be a shift from spoken to written linguistic skills. This will provide you with new academic, narrative and journalistic approaches to studying a language.
All of this is supported through four contact hours of teaching a week. You'll attend a two-hour seminar to practise listening and speaking. A one-hour lecture each week will teach you grammar and language functions. Reading practice, grammar exercises and character learning is covered in a weekly one-hour seminar. By the end of this module you should reach the B1-B2 Level of the CEFR (Common European Framework of Reference for Languages).
It's not necessary to have studied the Part I or Chinese Language 2 module first. However you must have reached a CEFR (Common European Framework of Reference for Languages) A2-B1 level of Chinese proficiency.
Chinese modern culture is profoundly influenced by its historical, commercial and philosophical tradition. This module will provide you with a general knowledge of the key aspects of Chinese socioeconomic expansion and its role in international relations.
From Daoism and Confucian philosophy to political strategy, from dining etiquette to indirect and impersonal communication, Chinese culture has evolved over thousands of years. We'll concentrate on China's economic and socio-political developments and intercultural communication. We will dive deep into the Chinese way of thinking and living.
An hour-long lecture and a one-hour seminar each week will present you with (inter-) cultural, political and historical knowledge. This will be invaluable if you're thinking about a career that will intersect with China and Chinese institutions.
This module includes authentic texts only slightly adapted from the originals, with a special focus on contemporary Chinese society and institutions. You will learn how to communicate comprehensively and systematically using the appropriate expressions and language norms in the right context.
You'll develop your skills in understanding and joining political, academic and journalistic discussions using advanced Chinese language skills. You'll be able to translate between English and Chinese and develop an idiomatic style of formal writing.
All of this is supported through four contact hours of teaching a week. You'll attend a two-hour seminar to practise listening and speaking. A one-hour lecture each week will teach you grammar and language functions. Reading and writing practice is covered in a weekly one-hour seminar.
It's not necessary to have studied the Part I, Chinese Language 2 or 3 modules before this. However you must have reached a CEFR (Common European Framework of Reference for Languages) B1-B2 level of Chinese proficiency.
As well as language and subject related skills, a degree in languages develops rich interpersonal, intercultural, cognitive and transferable skills that can be utilised across a variety of careers. Our graduates have found work in a wide variety of areas such as accountancy, IT, business development, civil service, events management, finance, journalism, publishing, research and sales, as well as teaching and translating both in the UK and abroad.
For the last ten years, languages graduates from Lancaster have been in the top ten universities in the country in terms of their employability with Iberian Languages ranking 1st for graduate prospects in the Complete University Guide 2017.
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A Primer on Chinese Pagodas: The Vajra Throne Pagoda 金剛寶座塔
Leave a comment researchtravelWoodenfish
By Peter Romaskiewicz
[Click here for the previous post on pagoda history]
Architecturally, East Asian pagodas are far more diverse than one might assume. Because of this diversity, Chinese art historians have developed several typologies to sort the varied pagoda structures. One categorization, based on architectural style, includes the following six types [depicted left to right below]:
Overturned Bowl Style Pagoda 覆缽式塔
Multistory Pavilion Style Pagoda 樓閣式塔
Tiered Eave Style Pagoda 簷式塔
Single Story Style Pagoda 單層式塔 [=亭閣式塔?]
Tibetan Style Pagoda 藏式塔
Vajra Throne Style Pagoda 金剛寶座式塔[1]
Illustrations of the Six-fold typology of Chinese pagodas (from FGS Encyclopedia of Buddhist Arts)
Of these, the first is the classical Indian stūpa, whose round body appears as if a begging bowl has been overturned. The next two have the iconic pagoda profile of a multistory tower accentuated by jutting eaves. The distinction is that the latter is not a true multistory structure because it cannot be ascended via an internal stairway. The fourth type consists of just a single story, while the fifth type is a later evolution of the Indic style, with a elevated and smaller circumference of the dome element. The last type, the Vajra Throne Style Pagoda, is a stylized version of the Mahābodhi Temple constructed on the site of Buddha's enlightenment (i.e the Vajra seat or throne) in Bodh Gāya, India. This post will focus on this last type of pagoda which is arguably the most distinctive of the six types noted above, being comprised of five spires atop a square foundation.
Vajra Throne Pagoda 金剛寶座塔 at Zhenjue Temple 真覺寺 in Beijing
The Mahābodhi Temple in India was likely constructed in the late sixth or early seventh century. This dating is suggested based on the reports of two Chinese pilgrims who visited the site. Faxian 法顯 (337-c. 422), who visited Bodh Gāya in the early fifth century, made no mention of the large structure, while Xuanzang 玄奘 (602-664), who travelled in the middle of the seventh century, comments on the precise measurements of the temple he found which coincides with the temple that remains there today.[2] An early Chinese representation of the Mahābodhi Temple is found in the murals of the Buddhist caves of Dunhuang 敦煌 in western China.[3] Eventually, several Mahābodhi Temple-inspired buildings were constructed in China, mostly during the Ming Dynasty (1368-1644).[4] The most important, completed in 1473, was built at Zhenjue Temple 真覺寺 in Beijing.
This last site is also called the Temple of the Five Pagodas 五塔寺 due to the appearance of the Mahābodhi Temple-inspired building which incorporates five pagodas into its architecture. The Chinese pagoda is named the Vajra Throne Pagoda 金剛寶座塔 after the traditional site where the Buddha was enlightened under the Bodhi Tree in Bodh Gāya, called the vajrāsana (Vajra Throne). Construction of the pagoda at Zhenjue Temple began during the reign of the Ming Emperor Yongle (r.1402-24) when a Buddhist monk named *Paṇḍita [?] 班迪達 from the "Western Regions" donated five Buddhist statues which the emperor instructed were to be housed in a special structure. He ordered that a pagoda be built that matched the architectural plans of the building found at the site of the vajrāsana. A horizontal plaque above the main entrance still reads "Imperially Constructed Vajra Throne Pagoda" 敕建金剛寶座塔. The pagoda was renovated during the Qing Dynasty, but is believed to retain some of the original structural elements. The square base of the Vajra Throne Pagoda is approximately 19 meters by 16 meters, and is about 8 meters high. This functions as a pedestal for five pagoda structures, each for one of the donated statues, of which the tallest central pagoda adds another 10 meters to the total height. These individual pagodas are each of the square, multi-eaved type. In addition a round terrace is placed in the center front, covering the top of a stairway that grants access to the top of the structure.
The sides of the main structure are ornately decorated with Buddhist images and symbols. Most of the space is covered by five horizontal registers which depict various buddhas.
* This post was written as an addendum to the lecture I gave on Sacred Buddhist Architecture at the 2018 Woodenfish.
[1] This is based on the Encyclopedia of Buddhist Arts published by Foguang Shan, found here. Other typologies may include the Road Spanning Style Pagoda 過街式塔, Sutra Pillar Style Pagoda 經幢式塔, and Flower Pagoda 華塔, among others. Some consider the Tibetan Style Pagoda a subtype of the Overturned Bowl Style Pagoda. Moreover, it seems the Pavillion Style Pagoda 亭閣式塔 is another name for the Single Story Style Pagoda, though I need to revisit this.
[2] Faxian arrived in Bodh Gāya in 409 and noted that a stūpa was found there, presumably an early structure built on the site. Xuanzang arrived in 637 and his descriptions and measurements match the building that remains today. See the comments in Cunningham 1892: 17-8.
[3] Found on the western wall of Cave 428.
[4] These include the pagodas of Biyun Temple 碧雲寺 in Beijing (completed in 1747), Xihuang Temple 西黃寺 in Beijing, Miaozhan Temple 妙湛寺 in Yunnan (built in 1458), Cideng Temple 慈燈寺 in Inner Mongolia (completed in 1732), and Zhenjue Temple discussed here. To this list can be added the Miaogao Pagoda 妙高塔 on Mt. Yuquan 玉泉山 in Beijing. It is worth noting that the famed Chinese architect Liang Sicheng, considered the Flower Pagoda to be a architectural precusor to the Vajra Throne Pagoda.
Abe, Stanley K. 2002. Ordinary Images. Chicago and London: University of Chicago Pres. [see esp. pp. 103-06, 123-66]
Di Luo. 2017. "A Grain of Sand: Miniatures as Sacred Repositories, Part I: The Longxingsi Sutra Case," [https://chinesearchitecture.wordpress.com/2017/05/29/a-grain-of-sand-miniatures-as-sacred-repositories-part-i-the-longxingsi-sutra-case/]
Di Luo. 2017. "A Grain of Sand: Miniatures as Sacred Repositories, Part II: The Huayansi Sutra Cabinets" [https://chinesearchitecture.wordpress.com/2017/05/30/a-grain-of-sand-miniatures-as-sacred-repositories-part-ii-the-huayansi-sutra-cabinets/]
Di Luo. 2017. "A Grain of Sand: Miniatures, Models, Simulacra" [https://chinesearchitecture.wordpress.com/2017/06/04/a-grain-of-sand-miniatures-models-simulacra/]
Fu, Xinian; Guo, Daiheng; Liu Xujie; Pan, Guxi; Qiao, Yun, Sun, Dazhang & Steinhardt, Nancy S. 2002. Chinese Architecture. New Haven: Yale University Press.
Kim, Youn-mi. 2017. "Virtual Pilgrimage and Virtual Geography: Power of Liao Miniature Pagodas (907–1125)," Religions, Vol. 8., pp. 1-29.
Le, Huu Phuoc. 2010. Buddhist Architecture. Lakeville, MN: Grafikol.
Ledderose, Lothar. 1980. "Chinese Prototypes of the Pagoda," in The Stupa, Its Religious, Historical, and Architectural Significance, ed. Anna Libera Dallapiccola and Stephanie Zingel-ave Lallement. Wiesbaden: Franz Steiner Verlag, pp. 238-45
Liang Ssu-ch'eng [Sicheng]. 1984. Chinese Architecture: A Pictorial History, ed. Wilma Fairbank. Cambridge: MIT Press. [see esp. p. 124]
Lin, Wei-cheng. 2016. "Performing Center in a Vertical Rise: Multilevel Pagodas in China's Medieval Period," ARS Orientalis, Vol. 46, pp. 100-34.
Neelis, Jason. 2011. Early Buddhist Transmission and Trade Networks: Mobility and Exchange within and beyond the Northwestern Borderlands of South Asia. Leiden: Brill.
Seckel, Dietrich. 1980. "Stupa Elements Surviving in Eastern Asian Pagodas," in The Stupa, Its Religious, Historical, and Architectural Significance, ed. Anna Libera Dallapiccola and Stephanie Zingel-ave Lallement. Wiesbaden: Franz Steiner Verlag.
Steinhardt, Nancy Shatzman. 1994. "Liao: An Architectural Tradition in the Making," Artibus Asiae, Vol. 54, No. 1/2, pp. 5-39.
Steinhardt, Nancy S. 1997. Liao Architecture. Honolulu: University of Hawaii Pres.
Steinhardt, Nancy S. 2011. "The Sixth Century in East Asian Architecture," Ars Orientalis, Vol. 41, pp. 27-71.
Steinhardt, Nancy S. 2014. Chinese Architecture in an Age of Turmoil, 200-600. Honolulu: University of Hawaii Press.


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To Help Latinos Age Well, We Need to Address Inequities
By Rebecca DeLaRosa • Sep 26, 2017
In California, the proportion of Latinos age 65 and older who live in poverty is 16 percent—twice the percentage of their white counterparts. Photo credit: Thinkstock.
Norma has been working since she was a teenager. She started working as a farmworker, then became a cannery worker and now works in childcare.
Despite decades of hard work—sometimes holding down two jobs—now at 60 years old, she is unsure if she "will ever be able to retire," said Norma, whose name has been changed.
Norma's story is illustrative of the challenges that Latino seniors face trying to afford retirement, health care, food and housing.
Latinos are the greatest growing demographic in California, comprising 75 percent of the population growth in the state, including 20 percent of adults 65 and over and a quarter of adults in the 50-64 age group.
Despite the economic and social challenges faced by the Latino community, Latinos often live long lives. However, our quality of life and health can be improved. The experience of our grandparents, parents and the Latino elder community is key in understanding California's most pressing health, social and economic justice challenges.
Norma, for example, has faced a lifetime of struggle trying to make ends meet.
"Sometimes I had to do two jobs and that was still not enough to pay for rent and food," she said. The Salinas resident decided to go back to school to get her daycare license so she could earn more; however, she was not able to finish her certification.
"My husband and I worked very hard because we wanted to buy a house where we could live with our children," Norma said. "We saved some money, but when we were ready to buy a house, our youngest daughter got very sick."
Because the family had savings, they didn't qualify for the state's low-income health program, Medi-Cal. But Norma and her husband's employers didn't provide health insurance, so the family "ended up paying for health care until we had no more savings," Norma said.
"That was the last time we were able to save that much money," she said.
Historically, Latinos as well as other elders of color, have had less access to various sources of retirement income, including pensions, savings, and access to safety net services. This has been the case because many of them grew up during a time of segregation.
Rebecca DeLaRosa is the director of legislative affairs for the Latino Coalition for a Healthy California.
Because of this, we see low rates of home ownership by Latinos. Home ownership has traditionally been seen as a way for families to grow wealth and have a good quality of life.
We can also see economic disparities in the savings of Medicare beneficiaries. White seniors save on average $91,950, black seniors save $12,350, and Latinos save $9,800.
One of the other factors leading to low retirement income for Latinos is that this demographic is over represented in low-income, physical labor jobs. Many times, these jobs provide no access to retirement savings plans that can supplement Social Security income. In addition, these jobs often put workers at risk of injury, reducing their earning potential.
As a result, in California, median annual income from Social Security for Latino seniors is $14,868. Surviving in California on such retirement income puts Latino seniors at great risk of homelessness and hunger.
In California, the proportion of Latinos age 65 and older who live in poverty is 16 percent—twice the percentage of their white counterparts. The disparities are even more perverse in regions like the Central Valley, where 46 percent of elder Latinos live in poverty compared to 39 percent of Latinos living in other parts of the state.
Food insecurity among Latino seniors is unfortunately too common. Latinos are about 3.5 times more likely than other Californians in their age group to suffer from food insecurity. Not to mention that over a third of Latinos (36 percent) in the Central Valley have limited access to food, compared to 21 percent of Latinos in other parts of the state.
We also know that fewer Latinos use services that can reduce their risk of homelessness and chronic disease, such as the Supplemental Nutrition Assistance Program and Supplemental Security Income, compared to other seniors, even though there is a great need.
In California, for example, only 18 percent of Latino seniors utilize one of the food assistance programs, CalFresh or Supplemental Nutrition Assistance Program. When it comes to the Supplemental Security Income, which helps adults with food, medication, housing and other necessities, only 6 percent of eligible Latino seniors are enrolled in the program.
Without accessing benefits, Latino seniors may need to continue working into old age or to retrain and seek jobs in a new sector. This poverty cycle and lack of access to health care preventive services and healthy food puts Latino seniors at risk for higher complication rates for preventable diseases, leading to low quality of life.
Having policies and programs in place that address the economic and social disparities of our Latino elders is critical for the well-being of everyone. In my next op ed, I will present solutions that can help our elders age well.
These solutions can help the many Latino seniors like Norma who struggle to make ends meet.
"I don't know if I will be able to live with my retirement payment," Norma said. "I am always looking for an opportunity to make extra money."
by Rebecca DeLaRosa, California Health Report

To Help Latinos Age Well, We Need to Address Inequities

by Rebecca DeLaRosa, California Health Report
September 26, 2017

Norma has been working since she was a teenager. She started working as a farmworker, then became a cannery worker and now works in childcare.

Despite decades of hard work—sometimes holding down two jobs—now at 60 years old, she is unsure if she "will ever be able to retire," said Norma, whose name has been changed.

Norma's story is illustrative of the challenges that Latino seniors face trying to afford retirement, health care, food and housing.

Latinos are the greatest growing demographic in California, comprising 75 percent of the population growth in the state, including 20 percent of adults 65 and over and a quarter of adults in the 50-64 age group.

Despite the economic and social challenges faced by the Latino community, Latinos often live long lives. However, our quality of life and health can be improved. The experience of our grandparents, parents and the Latino elder community is key in understanding California's most pressing health, social and economic justice challenges.

Norma, for example, has faced a lifetime of struggle trying to make ends meet.

"Sometimes I had to do two jobs and that was still not enough to pay for rent and food," she said. The Salinas resident decided to go back to school to get her daycare license so she could earn more; however, she was not able to finish her certification.

"My husband and I worked very hard because we wanted to buy a house where we could live with our children," Norma said. "We saved some money, but when we were ready to buy a house, our youngest daughter got very sick."

Because the family had savings, they didn't qualify for the state's low-income health program, Medi-Cal. But Norma and her husband's employers didn't provide health insurance, so the family "ended up paying for health care until we had no more savings," Norma said.

"That was the last time we were able to save that much money," she said.

Historically, Latinos as well as other elders of color, have had less access to various sources of retirement income, including pensions, savings, and access to safety net services. This has been the case because many of them grew up during a time of segregation.

Because of this, we see low rates of home ownership by Latinos. Home ownership has traditionally been seen as a way for families to grow wealth and have a good quality of life.

We can also see economic disparities in the savings of Medicare beneficiaries. White seniors save on average $91,950, black seniors save $12,350, and Latinos save $9,800.

One of the other factors leading to low retirement income for Latinos is that this demographic is over represented in low-income, physical labor jobs. Many times, these jobs provide no access to retirement savings plans that can supplement Social Security income. In addition, these jobs often put workers at risk of injury, reducing their earning potential.

As a result, in California, median annual income from Social Security for Latino seniors is $14,868. Surviving in California on such retirement income puts Latino seniors at great risk of homelessness and hunger.

In California, the proportion of Latinos age 65 and older who live in poverty is 16 percent—twice the percentage of their white counterparts. The disparities are even more perverse in regions like the Central Valley, where 46 percent of elder Latinos live in poverty compared to 39 percent of Latinos living in other parts of the state.

Food insecurity among Latino seniors is unfortunately too common. Latinos are about 3.5 times more likely than other Californians in their age group to suffer from food insecurity. Not to mention that over a third of Latinos (36 percent) in the Central Valley have limited access to food, compared to 21 percent of Latinos in other parts of the state.

We also know that fewer Latinos use services that can reduce their risk of homelessness and chronic disease, such as the Supplemental Nutrition Assistance Program and Supplemental Security Income, compared to other seniors, even though there is a great need.

In California, for example, only 18 percent of Latino seniors utilize one of the food assistance programs, CalFresh or Supplemental Nutrition Assistance Program. When it comes to the Supplemental Security Income, which helps adults with food, medication, housing and other necessities, only 6 percent of eligible Latino seniors are enrolled in the program.

Without accessing benefits, Latino seniors may need to continue working into old age or to retrain and seek jobs in a new sector. This poverty cycle and lack of access to health care preventive services and healthy food puts Latino seniors at risk for higher complication rates for preventable diseases, leading to low quality of life.

Having policies and programs in place that address the economic and social disparities of our Latino elders is critical for the well-being of everyone. In my next op ed, I will present solutions that can help our elders age well.

These solutions can help the many Latino seniors like Norma who struggle to make ends meet.

"I don't know if I will be able to live with my retirement payment," Norma said. "I am always looking for an opportunity to make extra money."

Rebecca DeLaRosa is the director of legislative affairs for the Latino Coalition for a Healthy California.

This article first appeared on California Health Report and is republished here under a Creative Commons license.
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