Cell‐penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myoblasts

Kwak, Kyu Bong; Kambayashi, Jun-ichi; Kang, M. S.; Ha, Doo Bong; Chung, Chin Ha

doi:10.1016/0014-5793(93)81468-f

Cited by 52 publications

(28 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Filamin repeats near hinge-1 have been identified previously as the sites of interaction for androgen (28) and dopamine D 2 (29) receptors and tumor necrosis factor receptor-associated factor 2 (30), indicating a general role for this region in anchoring membrane-associated signaling proteins to the actin cytoskeleton. The hinge-1 region is also recognized for its susceptibility to cleavage by calcium-activated proteases such as calpain, which prevents filamin from cross-linking actin and alters the stability of the cytoskeletal network (31)(32)(33). Nephrocystin binding could alter the conformation of the hinge-1 region and affect cleavage, though we have been unable to show an effect of nephrocystin binding on filamin degradation by calpain in vitro.…”

Section: Discussionmentioning

confidence: 93%

Nephrocystin-conserved Domains Involved in Targeting to Epithelial Cell-Cell Junctions, Interaction with Filamins, and Establishing Cell Polarity

Donaldson¹,

Dise²,

Ritchie³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Nephrocystin is the protein product of the gene mutated in juvenile nephronophthisis, an autosomal recessive cystic kidney disease afflicting children and young adults. Because the normal cellular function of nephrocystin is largely unknown, the molecular defects underlying disease pathogenesis remain obscure. Analysis of nephrocystin amino acid sequences from human and other species revealed three distinct conserved domains including Src homology 3 and coil-coil domains in the N-terminal region, as well as a large highly conserved C-terminal region bearing no obvious homology to other proteins and hence referred to as the "nephrocystin homology domain" (NHD). The objective of this study was to gain insight into nephrocystin function by defining functional properties of the conserved domains. We analyzed a series of nephrocystin deletion mutants expressed in Madin-Darby canine kidney and COS-7 cells. This analysis revealed previously unrecognized functional attributes of the NHD, including abilities to promote both self-association and epithelial cell-cell junctional targeting. We further observed that Madin-Darby canine kidney cell lines stably expressing a nephrocystin mutant with a deletion of the Src homology 3 domain have reduced ability to establish tight junctions as measured by transepithelial electrical resistance. Finally, from a two-hybrid screen and coimmunoprecipitation studies we identified members of the filamin family of actin-binding proteins as having the capacity to interact with the NHD. These findings support a functional role for nephrocystin as a docking protein involved in organizing a protein complex to regulate the actin cytoskeleton at sites of epithelial cell-cell adhesion and further suggest that these properties are important for establishing epithelial cell polarity.

show abstract

Section: Discussionmentioning

confidence: 93%

Nephrocystin-conserved Domains Involved in Targeting to Epithelial Cell-Cell Junctions, Interaction with Filamins, and Establishing Cell Polarity

Donaldson¹,

Dise²,

Ritchie³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…(ii) Inhibitors of protease activity some of which are specific for calpain (e.g., calpain antibody, human calpastatin peptide, endogenous rabbit calpastatin) inhibit seal formation in crayfish MGAs. (iii) Calpain is an endogenous protease in squid GAs (5), other axons (including the crayfish MGA) (4,6,8,10,20), and many other cell types (14)(15)(16)(17)(18)(19). (While other proteases may induce sealing in Ca 2ϩ -free salines in crayfish MGAs (this report) and in mammalian axons (11), these proteases are not known to be endogenous to these axons.)…”

Section: Resultsmentioning

confidence: 99%

Calpain activity promotes the sealing of severed giant axons

Godell

Smyers

Eddleman

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injuryinduced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca 2؉ -free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.The rapid (within 60 min) repair of unmyelinated crayfish medial giant axons (MGAs) (1) and myelinated earthworm MGAs (2) is associated with the accumulation of vesicles and other membranous material at the cut axonal ends. In contrast, squid giant axons (GAs) do not seal, even though injuryinduced vesicles form after transection and accumulate at cut axonal ends (3). Calpain (4) is a calcium-activated neutral protease endogenous to squid GAs (5), other axons (4), and other cell types (4, 6) that could enhance or inhibit one or more stages (vesicle formation, movement, aggregation, membrane fusion, etc.) of the sealing process. Calpain and other proteases degrade cytoskeletal proteins such as neurofilaments (4,6,7,8), which help maintain axonal diameter and shape (9). Such degradation might produce a complete or partial collapse of the cut end (10, 11), thereby inducing sealing by enhancing the interactions between vesicles that form a seal by themselves and͞or that form a continuous membrane at the cut end. (In this paper, the term ''complete constriction or complete collapse'' is used to describe 100% closure of a cut axonal end; the term ''partial constriction or partial collapse'' is used to describe any constriction that leaves an opening at the cut end.) Alternatively, calpain or other proteases might inhibit sealing by repressing the formation of vesicles or a continuous membrane or by degrading cytoskeletal proteins (microtubules, actin, molecular motors, etc.), which might affect the movement of injury-induced or other vesicles to the cut axonal end.We examined the effects of calpain and other proteases on the sealing of two giant axons: the crayfish MGA, which normally seals after severance in physiological saline (see preceding paper, ref. 1), and the squid GA, which normally does not seal after severance in physiological saline (2). [GAs (2) and MGAs (1) transected in Ca 2ϩ -free salines do not seal.] According to our electrophysiological measures (decay of injury current, I i , and resting membrane potential, V m ) and optical measures (dye exclusion), we find that exogenous calpain, but not chymotrypsin, i...

show abstract

“…Exogenously added calpain II was found to promote myoblast fusion (40). Studies using calpain inhibitors also showed that inhibition of the endogenous calpain can suppress myogenesis (14,41). Although possible roles of calpain II in muscle growth and differentiation have been postulated (6,42), the biochemical mechanism underlying its myogenic function is still unclear.…”

Section: Discussionmentioning

confidence: 99%

Proteolytic Regulation of the Zinc Finger Transcription Factor YY1, a Repressor of Muscle-restricted Gene Expression

Walowitz

Bradley

Chen

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Regulated proteolysis has been postulated to be critical for proper control of cell functions. Muscle development, in particular, involves a great deal of structural adaptation and remodeling mediated by proteases. The transcription factor YY1 represses muscle-restricted expression of the sarcomeric ␣-actin genes. Consistent with this repressor function of YY1, the nuclear regulator is down-regulated at the protein level during skeletal as well as cardiac muscle cell differentiation. However, the YY1 message remains relatively unaltered throughout the myoblast-myotube transition, implicating a post-translational regulatory mechanism. We show that YY1 can be a substrate for cleavage by the calciumactivated neutral protease calpain II (m-calpain) and the 26 S proteasome. The calcium ionophore A23187 destabilized YY1 in cultured myoblasts, and the decrease in YY1 protein levels could be prevented by calpain inhibitor II and calpeptin. Treatment with the proteasome inhibitors MG132 and lactacystin resulted in the stabilization of YY1 protein, which is consistent with the finding that YY1 is readily polyubiquitinated in reticulocyte lysates. We further show that proteolytic targeting by calpain II and the proteasome involves different structural elements of YY1. This study thus illustrates two proteolytic pathways through which the transcriptional regulator can be differentially targeted under different cell growth conditions. Many cellular processes are known to be controlled by shortlived proteins, including products of the proto-oncogenes, cell cycle regulators, and developmentally regulated transcription factors (1-3). The fast turnover of these regulatory proteins reflects a metabolic requirement for rapidly changing their concentrations and is presumably mediated by a complex interplay among various proteases and protease inhibitors. Selective degradation of transcriptional activators and repressors, in particular, may provide efficient regulatory mechanisms contributing to the rapid shut-off and turn-on of gene activity, respectively. The recent study of the pleiotropic transcription factor NF-B has revealed that the ubiquitinproteasome pathway can function not only in the complete degradation of proteins but also in the regulated processing of precursors into active transcription factors (4, 5). Calpains represent the other major class of nonlysosomal proteases functioning in a calcium-dependent fashion (6, 7). Interestingly, both the proteasome and calpains have been found to play a regulatory role in the function and/or stability of c-Fos and the tumor suppressor protein p53 (8 -11). Thus, rapid degradation or processing of specific transcription factors can underlie a wide range of dynamic cellular and developmental processes.Muscle development involves a great deal of structural adaptation and remodeling mediated by induced protein synthesis and degradation (12)(13)(14). Although protein turnover must be highly selective if it is to be developmentally useful, little is known concerning the regulatory mech...

show abstract

Cell‐penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myoblasts

Cited by 52 publications

References 30 publications

Nephrocystin-conserved Domains Involved in Targeting to Epithelial Cell-Cell Junctions, Interaction with Filamins, and Establishing Cell Polarity

Nephrocystin-conserved Domains Involved in Targeting to Epithelial Cell-Cell Junctions, Interaction with Filamins, and Establishing Cell Polarity

Calpain activity promotes the sealing of severed giant axons

Proteolytic Regulation of the Zinc Finger Transcription Factor YY1, a Repressor of Muscle-restricted Gene Expression

Contact Info

Product

Resources

About