This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell dea...

Ischemic Cell Death in Brain Neurons

The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

The Calpain System

Charge, Sophie B. P., and Michael A. Rudnicki. Cellular and Molecular Regulation of Muscle Regeneration. Physiol Rev 84: 209–238, 2004; 10.1152/physrev.00019.2003.—Under normal circumstances, mamma...

Cellular and Molecular Regulation of Muscle Regeneration

Repeated, intense use of muscles leads to a decline in performance known as muscle fatigue. Many muscle properties change during fatigue including the action potential, extracellular and intracellular ions, and many intracellular metabolites. A range of mechanisms have been identified that contribute to the decline of performance. The traditional explanation, accumulation of intracellular lactate and hydrogen ions causing impaired function of the contractile proteins, is probably of limited importance in mammals. Alternative explanations that will be considered are the effects of ionic changes on the action potential, failure of SR Ca2+ release by various mechanisms, and the effects of reactive oxygen species. Many different activities lead to fatigue, and an important challenge is to identify the various mechanisms that contribute under different circumstances. Most of the mechanistic studies of fatigue are on isolated animal tissues, and another major challenge is to use the knowledge generated in these studies to identify the mechanisms of fatigue in intact animals and particularly in human diseases.

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Skeletal Muscle Fatigue: Cellular Mechanisms

The first suggestion that physical exercise results in free radical-mediated damage to tissues appeared in 1978, and the past three decades have resulted in a large growth of knowledge regarding exercise and oxidative stress. Although the sources of oxidant production during exercise continue to be debated, it is now well established that both resting and contracting skeletal muscles produce reactive oxygen species and reactive nitrogen species. Importantly, intense and prolonged exercise can result in oxidative damage to both proteins and lipids in the contracting myocytes. Furthermore, oxidants can modulate a number of cell signaling pathways and regulate the expression of multiple genes in eukaryotic cells. This oxidant-mediated change in gene expression involves changes at transcriptional, mRNA stability, and signal transduction levels. Furthermore, numerous products associated with oxidant-modulated genes have been identified and include antioxidant enzymes, stress proteins, DNA repair proteins, and mitochondrial electron transport proteins. Interestingly, low and physiological levels of reactive oxygen species are required for normal force production in skeletal muscle, but high levels of reactive oxygen species promote contractile dysfunction resulting in muscle weakness and fatigue. Ongoing research continues to probe the mechanisms by which oxidants influence skeletal muscle contractile properties and to explore interventions capable of protecting muscle from oxidant-mediated dysfunction.

/pdf/exercise-induced-oxidative-stress-cellular-mechanisms-and-4a18810890.pdf

Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production

Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1-hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca2+.

Interaction of calpastatin with calpain: a review.

Changes in activity and protein status of μ-calpain, m-calpain, and calpastatin in bovine semimembranosus muscle during the first 7 d of postmortem storage were monitored by using assays of proteolytic activity, SDS-polyacrylamide gel electrophoresis, and Western blot analysis. Extractable m-calpain activity changed slightly during the first 7 d after death (decreased to 63% of at-death activity after 7 d), whereas extractable calpastatin activity decreased substantially (to 60% of at-death activity after 1 d and to 30% of at-death activity after 7 d of postmortem storage) during this period. Extractable μ-calpain activity also decreased rapidly (to 20% of at-death activity at 1 d and to less than 4% of its at-death activity at 7 d after death) during postmortem storage. Western blot analysis showed that the 80-kDa subunit of m-calpain remained undegraded during the first 7 d after death but that the 125- to 130-kDa calpastatin polypeptide was gone entirely at 7 d after death. Hence, the calpastatin activity remaining at 7 d originates from calpastatin polypeptides that are 42 kDa or smaller. The 80-kDa μ-calpain subunit was almost entirely in the 76-kDa autolyzed form at 7 d after death; this form is proteolytically active in in vitro systems, and it is unclear why the postmortem, autolyzed μ-calpain is not active. Over 50% of total muscle μ-calpain is tightly bound to myofibrils 7 d after death; this μ-calpain is also nearly inactive proteolytically. Unless postmortem muscle contains some factor that enables μ-calpain in this muscle to be proteolytically active, it is not clear whether μ-calpain could be responsible for any appreciable postmortem myofibrillar proteolysis.

Changes in the calpains and calpastatin during postmortem storage of bovine muscle.

RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA

/pdf/intrinsically-disordered-rgg-rg-domains-mediate-degenerate-3891yg25bj.pdf

Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding

The first protein of a group of proteins now identified as belonging to the calpain system was purified in 1976. The calpain system presently is known to be constituted of three well-characterized proteins; several lesser studied proteins that have been isolated from invertebrates; and 10 mRNAs, two each in Drosophila and C. elegans and six in vertebrates, that encode proteins, which, based on sequence homology, belong to the calpain family. The three well-characterized proteins in the calpain family include two Ca2+-dependent proteolytic enzymes, µ-calpain and m-calpain, and a protein, calpastatin, that has no known activity other than to inhibit the two calpains. A substantial amount of experimental evidence accumulated during the past 25 yr has shown that the calpain system has an important role both in rate of skeletal muscle growth and in rate and extent of postmortem tenderization. Calpastatin seems to be the variable component of the calpain system, and skeletal muscle calpastatin activity is highl...

Valery F. Thompson

Papers

Interaction of calpastatin with calpain: a review.

Changes in the calpains and calpastatin during postmortem storage of bovine muscle.

Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding

The calpain system and skeletal muscle growth

Comparison of the autolyzed and unautolyzed forms of μ- and m-calpain from bovine skeletal muscle