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Results for “Protein Isoforms”

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PubMed1998

The human UNP locus at 3p21.31 encodes two tissue-selective, cytoplasmic isoforms with deubiquitinating activity that have reduced expression in small cell lung carcinoma cell lines.

The human Unp gene at 3p21.3 has sequence similarity to ubiquitin proteases and has been suggested to play a role in carcinogenesis of the lung (Gray et al., 1995). To investigate this possibility, we isolated cDNAs from several human tissue libraries and found evidence for two major isoforms, encoding proteins predicted to either contain an internal 47 amino acid segment or not. Both are functional in deubiquitination assays, and mutation of a critical conserved cysteine residue to alanine abolished activity. Unp specifies two closely-migrating transcripts whose relative abundance varies among human adult tissues. Antibodies specific to UNP confirm the presence of at least two endogenous protein isoforms of approximately 105-110 kDa in cell lysates, as predicted from the cDNA sequences. Cellular fractionation and immunocytochemistry revealed UNP expression localized primarily in the cytoplasm. When we examined a panel of lung-derived cell lines for both UNP mRNA and protein expression, we found reduced levels of UNP protein in all four small cell lung carcinoma cell lines tested. These findings directly contradict and offer alternative interpretations to a number of previously published observations on Unp.

Adult↗

PubMed1992

Localization and differential expression of two isoforms of the tight junction protein ZO-1.

ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoforms contribute to tight junction diversity in different epithelia.

Amino Acid Sequence↗

PubMed1995

Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. Possible molecular mechanisms.

We have previously reported that two closely related protein kinase C (PKC) isoforms, PKC alpha and PKC beta I, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W. O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKC beta I lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKC alpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKC alpha and PKC beta I. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKC beta I differed from those overexpressing PKC alpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKC beta I may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.

Animals↗

PubMed1998

Phorbol ester-induced G1 arrest in BALB/MK-2 mouse keratinocytes is mediated by delta and eta isoforms of protein kinase C.

We investigated the possible negative regulation of the cell cycle by protein kinase C (PKC) isoforms in synchronously grown BALB/MK-2 mouse keratinocytes, in which PKC isoforms were overexpressed by using the adenovirus vector Ax. Cells at the G1/S boundary of the cell cycle were the most sensitive to the inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC agonist, resulting in G1 arrest. TPA-induced inhibition of DNA synthesis was augmented by overexpression of the eta and delta isoforms, but rescued by the dominant-negative and antisense eta isoforms. In contrast, the alpha and zeta isoforms showed no effect on DNA synthesis with or without TPA treatment. Immunoblotting indicated cell cycle-dependent expression of the eta isoform, being highest in cells at the G1/S boundary. The present study provides evidence that the eta and delta isoforms of PKC are involved in negative regulation of cell cycle at the G1/S boundary in mouse keratinocytes.

Adenoviridae↗

PubMed1997

Vertebrates have conserved capping protein alpha isoforms with specific expression patterns.

Capping protein (CP), a ubiquitous actin binding protein composed of an alpha and a beta subunit, is important for actin assembly and cell motility. Lower organisms have one gene and one isoform of each subunit. Chickens have two very similar alpha-subunit isoforms. To determine if vertebrates in general contain multiple alpha isoforms and if those alpha isoforms have conserved sequences, we isolated and analyzed alpha subunit cDNA's in mice and humans. Both mice and humans also have two alpha isoforms. Phylogenetic analysis of the alpha isoform sequences reveals that vertebrates have two highly conserved subfamilies, alpha1 and alpha2. The alpha1 and alpha2 subfamilies are very similar to each other but can be defined and distinguished from each other by a small number of key amino acid residues. In addition, 3' untranslated cDNA sequences are conserved within the isoform subfamilies. To investigate the function of the alpha isoforms, we examined their expression in mouse cells and tissues. Endothelial cells contain only the alpha2 isoform, and erythrocytes contain almost exclusively the alpha1 isoform. Most tissues have both alpha1 and alpha2 isoforms but the ratio of alpha1:alpha2 varies widely. Together, these findings support the hypothesis that the CP alpha isoforms have conserved, unique and essential roles in vertebrates.

Actin Depolymerizing Factors↗

PubMed2002

CCAAT/enhancer binding proteins in normal mammary development and breast cancer.

CCAAT/enhancer binding proteins (C/EBPs) are a family of leucine zipper, transcription factors that bind to DNA as homodimers and heterodimers. They regulate cellular proliferation, differentiation and apoptosis in the mammary gland. Multiple protein isoforms, including truncated, dominant negatives, are generated by translation of the C/EBPbeta transcript or via proteolytic cleavage of the full-length C/EBPbeta protein. Gene deletion of individual C/EBP family members has demonstrated an essential role for C/EBPbeta in normal mammary development, while transgenic and overexpression studies provide evidence that the dominant-negative C/EBPbeta-liver-enriched inhibitory protein isoform induces proliferation in mammary epithelial cells. Mounting evidence suggests that alterations in the ratio of the C/EBPbeta-liver-enriched inhibitory protein isoform and the C/EBPbeta-liver-enriched activating protein isoform may play a role in the development of breast cancer. This review will consequently focus on C/EBP actions in normal mammary development and on the emerging data that supports a role in breast cancer.

Animals↗

PubMed1988

Expression of specific isoforms of protein 4.1 in erythroid and non-erythroid tissues.

Protein 4.1 in red cells is an important submembrane linking protein that binds to spectrin actin complexes at one end of its structure and to transmembrane proteins, such as glycophorin, at the other. Protein 4.1 thus contributes to the strength and flexibility of the erythrocyte membrane, a fact dramatically exemplified by the appearance of hereditary hemolytic anemias in patients with absent or abnormal protein 4.1. Recently, protein 4.1 forms have been discovered in many non-erythroid tissues. Their intracellular locations raise the possibility that these isoforms might have different functions. We have thus conducted comparative analysis of erythroid and non-erythroid protein 4.1 forms by cloning and sequencing erythroid and lymphoid protein 4.1 cDNAs. The lymphoid protein 4.1 isoforms exhibit at least five nucleotide sequence motifs that appear to be either inserted or deleted relative to the erythroid mRNA sequence by alternative splicing of a common mRNA precursor. One of these motifs, located within the spectrin-actin binding domain, is found only in erythroid cells and is specifically produced during erythroid cell maturation. The selective expression of this alternatively spliced mRNA during erythroid maturation implies the existence of a lineage specific splicing mechanism whose activity is triggered by terminal maturation. Two motifs alter the 5' untranslated region of the "prototypical" erythroid mRNA in such a way as to permit synthesis of a novel larger isoform. This form appears to localize preferentially in the nucleus. We thus conclude that a single gene gives rise to multiple protein 4.1 isoforms with potentially diverse locations and functions.

Base Sequence↗

PubMed1992

Ca(2+)-independent isoforms of protein kinase C differentially translocate in smooth muscle.

It is generally assumed that smooth muscle contraction is dependent on changes in intracellular Ca2+ concentration ([Ca2+]i); however, we have previously reported that alpha-agonist-induced contraction of aorta smooth muscle cells can occur in the absence of changes in [Ca2+]i [Collins, E. M., M. P. Walsh, and K. G. Morgan. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992]. The mechanism of this [Ca2+]i-independent contraction is controversial. We have now identified the Ca(2+)-independent protein kinase C (PKC) isoforms epsilon and zeta in ferret aorta and have used digital imaging microscopy to determine their subcellular distribution. At rest, epsilon-PKC is diffusely distributed in the cytosol, whereas zeta-PKC is concentrated in the perinuclear region; both isoforms are excluded from the nuclear space. Agonist stimulation causes a [Ca2+]i-independent translocation of epsilon-PKC to the surface membrane and of zeta-PKC to the intranuclear compartment. In comparison, ferret portal vein cells, which display a totally Ca(2+)-dependent agonist contraction, are lacking in epsilon-PKC but display perinuclear zeta-PKC, which translocates intranuclearly on activation. Thus the Ca(2+)-independent vascular contraction appears to be associated with plasmalemmal translocation of epsilon-PKC; in contrast, the intranuclear translocation of zeta-PKC may function in control of gene expression.

Animals↗

PubMed1995

Developmental and regional expression of multifunctional Ca2+/calmodulin-dependent protein kinase isoforms in rat brain.

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) participates in diverse calcium signaling pathways in neurons. The alpha- and beta-CaM kinase isoforms are neuron-specific and highly abundant in rat brain. The variable domain of CaM kinase is a potential site for the generation of isoform diversity by alternative spicing of its N- and/or C-terminal segments. We used specific PCR primers which span the variable domain of either alpha- or beta-CaM kinase and isolated three new isoforms from rat brain, namely alpha B-, beta e- and beta'e-CaM kinase. alpha beta-CaM kinase contains 11 amino acids, likely inserted by alternative splicing, at the C-terminal segment of the variable domain. This insertion introduces a nuclear localization signal (NLS) that targets alpha B-CaM kinase to the nucleus of transfected cells; alpha-CaM kinase is excluded from the nucleus. The mRNA and the protein corresponding to this isoform are detected only in the diencephalon/midbrain regions. We have also identified two alternatively spliced isoforms of beta-CaM kinase that lack the 24 amino acid sequence at the N-terminal segment of the variable domain. Alternative splicing of these two isoforms occurs with a three base pair shift of the 3'-splice site. Our analysis shows that these new beta-CaM kinase isoforms are expressed primarily in early developmental stages, and we therefore term them beta e - (embryonic) and beta' e-CaM kinase. Recombinant alpha B-, beta e and beta' e-CaM kinase expressed in COS-7 cells exhibit characteristic Ca2+/calmodulin-dependent protein kinase activity and autophosphorylation.

Aging↗

PubMed2006

A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum.

Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.

Animals↗

PubMed2005

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability.

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Caco-2 Cells↗

PubMed2000

Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung diseases.

Recently, we published an analytical two-dimensional electrophoresis (2-DE) protein map of human bronchoalveolar lavage fluid (BALF) using a pool of BALFs from various patients. In this report, the effect of lung disorders on the protein composition of the lung epithelial lining fluid was investigated by 2-DE of BALFs from individual patients with well-defined interstitial lung diseases: sarcoidosis, idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP), using improved experimental conditions. On these gels, about 600-1000 stained protein spots could be identified in a BALF sample containing 25 microg of protein, and our original human BALF protein database has, therefore, been considerably extended. Altogether, 429 protein spots corresponding to 66 different proteins (including isoforms, protein subunits and fragments) were identified by microsequence analysis and by matching with a human blood plasma 2-DE protein map available in the SWISS-2DPAGE database. A human 2-DE BALF database was established and is available on the World Wide Web (http://www.umh.ac.be/-biochim/proteomic.htm+ ++). The significance of the modifications observed between the different lung pathologies is discussed with the aim of understanding the mechanistic bases of lung disease pathogenesis and finding new potential lung markers of disorders.

Adult↗

PubMed2005

Length-dependent Ca(2+) activation in cardiac muscle: some remaining questions.

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank-Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca(2+) sensitivity. How sarcomere length modulates Ca(2+) sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca(2+) sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca(2+) sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin-myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca(2+) sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca(2+) sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca(2+) sensitivity.

Animals↗

PubMed2004

Genes and gene expression in the brain of the alcoholic.

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABAA receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate and gamma-aminobutyric acid (GABA) receptors to influence the severity of alcohol-induced brain damage. Cerebrocortical tissue was obtained at autopsy from alcoholics without alcohol-related disease, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABB2, EAAT2, and 5HTT genotypes did not divide alcoholic cases and controls on N-methyl-d-aspartate (NMDA) receptor parameters. In contrast, alcohol dehydrogenase (ADH)3 genotype interacted significantly with NMDA receptor efficacy and affinity in a region-specific manner. EAAT2 genotype interacted significantly with local GABAA receptor beta subunit mRNA expression, and GABB2 and DRD2B genotypes with beta subunit isoform protein expression. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.

Adult↗

PubMed2006

Annotating the human proteome: beyond establishing a parts list.

The completion of the human genome has shifted the attention from deciphering the sequence to the identification and characterisation of the functional components, including genes. Improved gene prediction algorithms, together with the existing transcript and protein information, have enabled the identification of most exons in a genome. Availability of the 'parts list' has fostered the development of experimental approaches to systematically interrogate gene function on the genome, transcriptome and proteome level. Studying gene function at the protein level is vital to the understanding of how cells perform their functions as variations in protein isoforms and protein quantity which may underlie a change in phenotype can often not be deduced from sequence or transcript level genomics experiments alone. Recent advancements in proteomics have afforded technologies capable of measuring protein expression, post-translational modifications of these proteins, their subcellular localisation and assembly into complexes and pathways. Although an enormous amount of data already exists on the function of many human proteins, much of it is scattered over multiple resources. Public domain databases are therefore required to manage and collate this information and present it to the user community in both a human and machine readable manner. Of special importance here is the integration of heterogeneous data to facilitate the creation of resources that go beyond a mere parts list.

Humans↗

PubMed2003

Neurosteroid modulation of GABAA receptors.

Certain metabolites of progesterone and deoxycorticosterone are established as potent and selective positive allosteric modulators of the gamma-aminobutyric acid type A (GABA(A)) receptor. Upon administration these steroids exhibit clear behavioural effects that include anxiolysis, sedation and analgesia, they are anticonvulsant and at high doses induce a state of general anaesthesia, a profile consistent with an action to enhance neuronal inhibition. Physiologically, peripherally synthesised pregnane steroids derived from endocrine glands such as the adrenals and ovaries function as hormones by crossing the blood brain barrier to influence neuronal signalling. However, the demonstration that certain neurons and glial cells within the central nervous system (CNS) can synthesize these steroids either de novo, or from peripherally derived progesterone, has led to the proposal that these steroids (neurosteroids) can additionally function in a paracrine manner, to locally influence GABAergic transmission. Steroid levels are known to change dynamically, for example in stress and during pregnancy. Given that GABA(A) receptors are ubiquitously expressed throughout the central nervous system, such changes in steroid levels would be predicted to cause a global enhancement of inhibitory neurotransmission throughout the brain, a scenario that would seem incompatible with a physiological role as a selective neuromodulator. Here, we will review emerging evidence that the GABA-modulatory actions of the pregnane steroids are highly selective, with their actions being brain region and indeed neuron dependent. Furthermore, the sensitivity of GABA(A) receptors is not static but can dynamically change. The molecular mechanisms underpinning this neuronal specificity will be discussed with particular emphasis being given to the role of GABA(A) receptor isoforms, protein phosphorylation and local steroid metabolism and synthesis.

Animals↗

PubMed2002

Changes in brain Na, K-ATPase isoform expression and enzymatic activity after aortic constriction.

The objective of the present study was to test the hypothesis that brain Na, K-ATPase expression and/or activity is altered following an increase in blood pressure produced by constriction of the abdominal aorta just proximal to the renal arteries. A suprarenal constriction (SRC) was made to conform to the diameter of a 19-gauge (19-G) or 20-gauge (20-G) needle, while in a sham-operated group (Sham) the aorta was exposed surgically but not constricted. Within 1 week of SRC, mean arterial pressure was increased and remained elevated at 4 weeks post surgery. At 1 week, whole-brain Na, K-ATPase mRNA levels were depressed for all isoforms (alpha1 approximately beta1>alpha2>alpha3). No changes were observed in the hypothalamus. At 4 weeks, the mRNA levels of all alpha isoforms were significantly increased in the whole brain and these changes were paralleled by an increase of alpha2 and alpha3 transcript in the hypothalamus. beta1 mRNA expression was increased in the hypothalamus only. The alpha-isoform protein expression generally changed in the same direction as mRNA changes at both 1 and 4 weeks, as did alpha1 enzyme activity at 1 week and the combined alpha2/alpha3 enzyme activities at 4 weeks. Since inhibition of brain Na, K-ATPase increases sympathetic nervous system (SNS) activity and blood pressure, the decreases in brain Na, K-ATPase expression and activity at 1 week post SRC may contribute to the hypertension during its developmental phase, while the increase in the alpha2/alpha3 brain expression and activity at 4 weeks may be a compensatory response to established hypertension.

Animals↗

PubMed2003

Regulation of proteolysis during reloading of the unweighted soleus muscle.

There is little information on the mechanisms responsible for muscle recovery following a catabolic condition. To address this point, we reloaded unweighted animals and investigated protein turnover during recovery from this highly catabolic state and the role of proteolysis in the reorganization of the soleus muscle. During early recovery (18 h of reloading) both muscle protein synthesis and breakdown were elevated (+65%, P<0.001 and +22%, P<0.05, respectively). However, only the activation of non-lysosomal and Ca(2+)-independent proteolysis was responsible for increased protein breakdown. Accordingly, mRNA levels for ubiquitin and 20S proteasome subunits C8 and C9 were markedly elevated (from +89 to +325%, P<0.03) and actively transcribed as shown by the analysis of polyribosomal profiles. In contrast, both cathepsin D and 14-kDa-ubiquitin conjugating enzyme E2 mRNA levels decreased, suggesting that the expression of such genes is an early marker of reversed muscle wasting. Following 7 days of reloading, protein synthesis was still elevated and there was no detectable change in protein breakdown rates. Accordingly, mRNA levels for all the proteolytic components tested were back to control values even though an accumulation of high molecular weight ubiquitin conjugates was still detectable. This suggests that soleus muscle remodeling was still going on. Taken together, our observations suggest that enhanced protein synthesis and breakdown are both necessary to recover from muscle atrophy and result in catch-up growth. The observed non-coordinate regulation of proteolytic systems is presumably required to target specific classes of substrates (atrophy-specific protein isoforms, damaged proteins) for replacement and/or elimination.

Animals↗