Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation.

Amino acid deprivation activates the amino acid response (AAR) pathway that enhances transcription of genes containing an amino acid response element (AARE). The present data reveal a quantitative difference in the response to deprivation of individual amino acids. The AAR leads to increased eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and ATF4 translation. When HepG2 cells were deprived of an individual essential amino acid, p-eIF2alpha and activating transcription factor 4 were increased, but the correlation was relatively weak. Complete amino acid starvation in either Earle's balanced salt solution or Krebs-Ringer bicarbonate buffer (KRB) resulted in activation of transcription driven by a SNAT2 genomic fragment that contained an AARE. However, for the KRB, a proportion of the transcription was AARE-independent suggesting that amino acid-independent mechanisms were responsible. Therefore, activation of AARE-driven transcription is triggered by a deficiency in any one of the essential amino acids, but the response is not uniform. Furthermore, caution must be exercised when using a medium completely devoid of amino acids.

Identification of cathepsin L as a differentially expressed message associated with skeletal muscle wasting.

Alteration of skeletal muscle protein breakdown is a hallmark of a set of pathologies, including sepsis, with negative consequences for recovery. The aim of the present study was to search for muscle markers associated with protein loss, which could help in predicting and understanding pathological wasting. With the use of differential display reverse transcription-PCR, we screened differentially expressed genes in muscle from septic rats in a long-lasting catabolic state. One clone was isolated, confirmed as being overexpressed in septic skeletal muscle and identified as encoding the lysosomal cysteine endopeptidase cathepsin L. Northern- and Western-blot analysis of cathepsin L in gastrocnemius or tibialis anterior muscles of septic rats confirmed an elevation (up to 3-fold) of both mRNA and protein levels as early as 2 days post-infection, and a further increase 6 days post-infection (up to 13-fold). At the same time, the increase in mRNAs encoding other lysosomal endopeptidases or components of the ubiquitin-proteasome pathway did not exceed 4-fold. Cathepsin L mRNA was also increased in tibialis anterior muscle of rats treated with the glucocorticoid analogue, dexamethasone, or rats bearing the Yoshida Sarcoma. The increase in cathepsin L mRNA was reduced by 40% when the tumour-bearing animals were treated with pentoxifylline, an inhibitor of tumour necrosis factor-alpha production. In conclusion, these results demonstrate a positive and direct correlation between cathepsin L mRNA and protein level and the intensity of proteolysis, and identify cathepsin L as an appropriate early marker of muscle wasting. Cathepsin L presumably participates in the pathological response leading to muscle loss, with glucocorticoids and tumour necrosis factor-alpha potentially being involved in the up-regulation of cathepsin L.

Leucine limitation induces autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through a mammalian target of rapamycin-independent signaling pathway.

Loss of muscle mass usually characterizes different pathologies (sepsis, cancer, trauma) and also occurs during normal aging. One reason for muscle wasting relates to a decrease in food intake. This study addressed the role of leucine as a regulator of protein breakdown in mouse C2C12 myotubes and aimed to determine which cellular responses regulate the process. Determination of the rate of protein breakdown indicated that leucine is one key regulator of this process in myotubes because starvation for this amino acid is responsible for 30-40% of the total increase generated by total amino acid starvation. Leucine restriction rapidly accelerates the rate of protein breakdown (+11 to 15% (p < 0.001) after 1 h of starvation) in a dose-dependent manner. By using various inhibitors, evidence is provided that acceleration of protein catabolism results mainly from an induction of autophagy, activation of lysosome-dependent proteolysis, without modification of mRNA levels encoding the lysosomal cathepsins B, L, or D. Those results suggest that autophagy is an essential cellular response for increasing protein breakdown in muscle following food deprivation. Induction of autophagy precedes a decrease in global protein synthesis (-20% to -30% (p < 0.001)) that occurs after 3 h of leucine starvation. Inhibition of the mammalian target of rapamycin (mTOR) activity does not abolish the effect of leucine starvation and the level of phosphorylated ribosomal S6 protein is not affected by leucine withdrawal. These latter data provide clear evidence that the mTOR signaling pathway is not involved in the mediation of leucine effects on both protein synthesis and degradation in C2C12 myotubes.

Glucose controls cathepsin expression in Ras-transformed fibroblasts.

Overexpression and altered trafficking of cathepsins have been associated with the malignant properties of tumors and transformed cells. A characteristic phenotype of transformed cells is also a profound deviation in their metabolism (aerobic glycolysis, glutaminolysis) which enables them to adapt to extreme nutritional conditions. However, whether the altered metabolism may change the expression of proteinases involved in malignancy has not been determined. Herein we present evidences in Kirsten-virus-transformed 3T3 fibroblasts (KBALB) that D-glucose selectively increases active forms of cathepsins L, B, and D, without altering other lysosomal nonproteolytic hydrolases (beta-D-glucosaminidase, acid phosphatase, beta-D-glucuronidase, and beta-D-galactosidase). D-Glucose did not modify mRNA levels for cathepsin B or L and did not affect secretion of pro-cathepsin L. However, D-glucose enhanced strongly the amount of the mature forms of cathepsins B and L, without altering their preferential distribution to light endosomal fractions. Induction by d-glucose of intracellular mature cathepsins B and L required a high growth density of KBALB cells and was reproduced in BALB/3T3 fibroblasts stably transfected with a constitutively activated form of Ras. d-Glucose induction of active cathepsins however was not observed in nontransformed BALB/3T3. D-Mannose, in contrast to nonmetabolized sugars (D-galactose, or L-glucose), caused a similar increase in lysosomal cathepsin activities in dense KBALB cells. The D-glucose analogue, 3-O-methyl-D-glucose, which is transported but not further metabolized, did not reproduce the d-glucose effects. Our findings indicate that, dependent on the nutrient supply and as a consequence of their altered metabolism, transformed cells may modulate the production of active proteinases implicated in malignant progression.

Developmental control of cathepsin B expression in bovine fetal muscles.

Expression of lysosomal cysteine proteinases was studied during fetal calf muscle development. The peptide cleaving activities of cathepsins B and L decreased strongly from 80 to 250 days of fetal age. This decrease in cathepsin activities occurred similarly in three muscles exhibiting different metabolic and contractile properties in adult animals. Cathepsin B from adult or fetal muscle revealed similar enzymatic properties, but presented a five- to sixfold lower concentration in adult muscle as indicated by active-site titration with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guani din o)butane. During fetal growth, decreases in muscle cathepsin B specific activity and active enzyme concentration were associated with a parallel drop of cathepsin B mRNA levels. Bovine cathepsin B is encoded by two different transcripts resulting from alternative polyadenylation [Mordier, S. B., Béchet, D. M., Roux, M. P., Obled, A., and Ferrara, M. (1995) Eur. J. Biochem. 229, 35-44]. As revealed by ribonuclease protection assays, the two mRNAs encoding cathepsin B declined similarly during fetal muscle growth. This study indicates that lysosomal proteinases in skeletal muscle are under developmental control. The decrease of muscle cathepsins during fetal development appears sufficient to account for the low levels of these enzymes in adult muscles. In fetuses, high activities of lysosomal cysteine proteinases might be important for remodeling muscles during early development.

Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B.

Expression of lysosomal cysteine proteinases was studied during fetal calf myoblast-myotube differentiation. Activities of cathepsin B and L, but not cathepsin H, increase during bovine myogenic differentiation. In fetal muscle, cathepsin B and L activities are 2-4-fold orders of magnitude lower than in cultured myoblasts. Active-site titrations of cathepsin B with E-64 nevertheless reveal similar concentrations of active cathepsin B in myoblasts and myotubes, but 5-6-fold lower concentrations in fetal muscle. To specify whether concentrations of cathepsin B are related to levels of cathepsin B transcript, a cDNA clone encoding bovine cathepsin B was isolated and liquid hybridizations were performed with 32P-riboprobes complementary to the mRNA. In agreement with active-site titrations, there is no difference in cathepsin B mRNA levels between cultured myoblasts and myotubes, but lower levels of mRNA are found in fetal muscle. Concentrations of active cathepsin B therefore reflect levels of cathepsin B mRNA. Kinetic studies revealed that the catalytic efficiency (kcat/Km) of cathepsin B is 2-3-fold higher in myotubes than in myoblasts. The increase in cathepsin B activity during calf myoblast-myotube differentiation is thus due to modifications of enzymatic properties, and not of enzyme concentrations. The different catalytic efficiency of cathepsin B in myotubes and myoblasts was related neither to modifications of mRNA size, as revealed by Northern blot analysis, nor to a different Mr of the active enzyme, as revealed by affinity labeling with benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2, but to limited differences in cathepsin B isozymes.

Cimaterol reduces cathepsin activities but has no anabolic effect in cultured myotubes.

The effect of the beta-adrenergic agonist cimaterol on bovine and chicken primary myotubes was assessed. Cimaterol at 10-100 nM concentrations reduced cathepsin B benzyloxy-carbonyl-Arg-Arg-4-methyl-7-coumarylamide hydrolyzing activity, as well as benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide hydrolysis, which is a substrate for both cathepsin B and cathepsin L. Maximum effect was observed after 6-16 h treatment. Cathepsin H Arg-4-methyl-7-coumarylamide hydrolyzing activity was low and not significantly affected by cimaterol treatment. Despite decreasing cathepsin activities, cimaterol also increased proteolysis rates but induced no detectable effect on protein synthesis rates. These observations suggest that beta-agonists, as a result of a direct action on muscle, can decrease cathepsin activities but that beta-agonist-induced muscle hypertrophy may not be due to a direct effect on muscle cells.

Purification and properties of different isoforms of bovine cathepsin B.

A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30,000) and cleaved (Mr 25,000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 microM and 52 s-1, and 125 microM and 75 s-1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.

Lysosomal proteinase-sensitive regions in fast and slow skeletal muscle myosins.

We investigated the limited proteolysis of fast and slow myosins purified from rabbit psoas major and semimembranosus proprius muscles, respectively, by the main lysosomal proteinases: cathepsins B, H, L, and D. In EDTA containing buffer, cathepsin D cleaved both myosins only at the rod-S1 junction leading to the formation of two S1 fragments of slightly higher Mr than the three forms obtained with chymotrypsin. On addition of MgCl2 instead of EDTA, myosin hydrolysis was markedly reduced. In contrast, irrespective of the presence of either MgCl2 or EDTA, cathepsin B hydrolysed both myosins into HMM and LMM. Cathepsin L digested myosins more extensively than cathepsins B and D and the main fragments generated were, in decreasing order of importance, rod, S1, S2, HMM, and LMM. In the incubation conditions tested, cathepsin H displayed nondetectable action on myosins. As fast and slow myosin digest patterns were compared, the main differences observed concerned the size of the proteolytic products and the rate of hydrolysis, which was about 4-fold higher for the fast than for the slow isoform. This appeared consistent whatever enzyme was considered.

[Action of muscular proteinases on fast and slow myosins. Relation with post-mortem proteolysis in muscles of variable contractility]. / Action des protéinases musculaires sur les myosines rapide et lente. Relation avec la protéolyse post-mortem dans des muscles de type contractile variable.

Sensitivity to proteolysis of fast and slow rabbit skeletal muscle myosins with cathepsins D, B, H and L and with calpains I and II was studied in various incubation conditions. Cathepsins D, B and L degraded both myosins and exhibited different specificities towards these proteins. Moreover, in each case slow myosin appeared to be less sensitive to proteolysis than fast myosin. This finding agrees well with the lower extent of protein hydrolysis observed during post-mortem ageing of meat in slow twitch pork muscles.

Comparative action of cathepsins D, B, H, L and of a new lysosomal cysteine proteinase on rabbit myofibrils.

Specific action of cathespins D, B, H, L, and of a new high Mr (molecular weight relative to hydrogen) cysteine proteinase, on rabbit muscle myofibrils was studied at pH 5·7 by following changes affecting their ATPase activities, their calcium sensitivity, their effect on the ultrastructure, as well as the electrophoretic pattern of the contractile proteins in the presence of SDS. With regard to the MgCa-enhanced ATPase activity, all these proteinases had a very similar effect. A decrease in this activity was thus noted concomitantly with a shift of the straight-line graph obtained when plotting the present acto-myosin ATPase activity versus KCl concentrations. Cathepsins D, B, L and the high Mr cysteine proteinase induced a decrease in both the calcium ATPase activity of myosin and the calcium sensitivity of myofibrils. On the contrary, the Mg-EGTA-dependent ATpase activity was increased. Except for cathepsin H, extensive hydrolysis of proteins occurred in myofibrils treated with each of the lysosomal proteinases tested. However, different specificities could be distinguished. On the one hand, cathepsins D and B affected mainly myofibrillar protein running above and below actin, respectively, on SDS-polyacrylamide gel electrophoresis; on the other hand, the high Mr cysteine proteinase exhibited broader specificity since most of the proteins were hydrolyzed irrespective of their Mr. Myofibrils incubated with cathepsins B and the high Mr cysteine proteinase showed ultrastructural modifications at the level of Z-line, M-bands and A-bands. Myofibrils treated with cathepsin D and cathepsin H appeared almost unaltered. On the basis of these characteristics, cathepsin H hardly affected myofibrils. These results provide evidence for the involvement of the lysosomal proteinases in the meat ageing process and are discussed in regard to the changes occurring at the myofibrillar level during conversion of muscle into meat.

Species variations amongst proteinases in liver lysosomes.

Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.

Ewingella americana gen.nov., sp.nov., a new Enterobacteriaceae isolated from clinical specimens.

We propose the name Ewingella gen.nov. for a new group in the Enterobacteriaceae. Ewingella is phenotypically distinct from all other groups of Enterobacteriaceae. The members of this genus are lipase- and deoxyribonuclease-negative; Voges-Proskauer-positive; lysine-, ornithine- and arginine-decarboxylase-negative; anaerogenic; they produce acid from glucose in the presence (and absence) of iodoacetate, but fail to produce acid from L-arabinose, melibiose, raffinose, D-sorbitol or sucrose. DNA-relatedness studies (S1-nuclease method) showed that the 10 Ewingella strains studied form a single DNA-hybridization group which is less than 21% related to other members of the Enterobacteriaceae. This single DNA-hybridization group is named Ewingella americana sp. nov. The type strain of E. americana is CDC 1468-78 (= ATCC 33852 = CIP 8194). Although the 10 strains of E. americana were isolated from clinical sources in the United States, the clinical significance of these organisms is not known.