Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.     

Mapping von Willebrand factor A domain binding sites on a snake venom metalloproteinase cysteine-rich domain

The PIII class of the snake venom metalloproteinases (SVMPS) are acknowledged to be one of the major hemorrhage producing toxins in crotalid venoms. This class of SVMPS are structurally distinguished by the presence of disintegrin-like and cysteine-rich domains carboxy to the metalloproteinase domain and thus share structural homology with many of the ADAMs proteins. It has been suggested that the presence of the carboxy domain are the key structural determinants for potent hemorrhagic activity in that they may serve to target the proteinases to specific key extracellular matrix and cell surface substrates for proteolysis leading to hemorrhage production at the capillaries. Following from previous studies in our laboratory in this investigation we scanned the cysteine-rich domain of the PIII hemorrhagic SVMP jararhagin using synthetic peptides in an attempt to identify regions which could bind to von Willebrand factor (vWF), a known binding partner for jararhagin. From these studies we identified two such peptide, Jar6 and Jar7 that could support binding to vWF as well as block the recombinant cysteine-rich domain of jararhagin binding to vWF. Using the coordinates for the recently solved crystal structure of the PIII SVMP VAP1, we modeled the structure of jararhagin and attempted to dock the modeled cysteine-rich structure of that protein to the A1 domain of vWF. These studies indicated that effective protein-protein interaction between the two ligands was possible and supported the data indicating that the Jar6 peptide was involved, whereas the Jar7 peptide was observed to be sterically blocked from interaction. In summary, our studies have identified a region on the cysteine-rich domain of a PIII SVMP that interacts with vWF and based on molecular modeling could be involving in the interaction of the cysteine-rich domain of the SVMP with the A1 domain of vWF thus serving to target the toxin to the protein for subsequent proteolytic degradation.     

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.     

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with alpha2beta1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of alpha2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.     

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40?318 AMU for DM40 and 42?373 and 43?010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS–PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.     

The reprolysin jararhagin,a snake venom metalloproteinase,functions as a fibrillar collagen agonist involved in fibroblast cell adhesion and signaling

The integrins alpha(2)beta(1) and alpha(1)beta(1) have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to alpha(2)beta(1) regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the alpha(2)beta(1) integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor alpha(2)beta(1) in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins.     

Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family.

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.     

Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).     

High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom

High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.     

Connexin 43 confers resistance to hydrogen peroxide-mediated apoptosis

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase-3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.     

The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases

Metalloproteinases (MPs) are Zn(+)-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications.     

The cysteine-rich domain of snake venom metalloproteinases is a ligand for von Willebrand factor A domains: role in substrate targeting

Snake venom metalloproteinases (SVMPs) are members of the Reprolysin family of metalloproteinases to which the ADAM (a disintegrin and metalloproteinase) proteins also belong. The disintegrin-like/cysteine-rich domains of the ADAMs have been implicated in their function. In the case of the SVMPs, we hypothesized that these domains could function to target the metalloproteinases to key extracellular matrix proteins or cell surface proteins. Initially we detected interaction of collagen XIV, a fibril-associated collagen with interrupted triple helices containing von Willebrand factor A (VWA) domains, with the PIII SVMP catrocollastatin. Next we investigated whether other VWA domain-containing matrix proteins could support the binding of PIII SVMPs. Using surface plasmon resonance, the PIII SVMP jararhagin and a recombinant cysteine-rich domain from a PIII SVMP were demonstrated to bind to collagen XIV, collagen XII, and matrilins 1, 3, and 4. Jararhagin was shown to cleave these proteins predominantly at sites localized at or near the VWA domains suggesting that it is the VWA domains to which the PIII SVMPs are binding via their cysteine-rich domain. In light of the fact that these extracellular matrix proteins function to stabilize matrix, targeting the SVMPs to these proteins followed by their specific cleavage could promote the destabilization of extracellular matrix and cell-matrix interactions and in the case of capillaries could contribute to their disruption and hemorrhage. Although there is only limited structural homology shared by the cysteine-rich domains of the PIII SVMPs and the ADAMs our results suggest an analogous function for the cysteine-rich domains in certain members of the expanded ADAM family of proteins to target them to VWA domain-containing proteins.     

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background

Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.

Methodology/Principal Findings

In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.

Conclusions/Significance

These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.     

The hallmarks of myotonic dystrophy type 1 muscle dysfunction

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3′ untranslated region (3′UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin–proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.     

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in “phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.     

The up-regulation of stromelysin-1 (MMP-3) in a spontaneously demyelinating transgenic mouse precedes onset of disease

The matrix metalloproteinases (MMPs) are a family of endoproteinases that degrade various components of the extracellular matrix and have been implicated in the pathogenesis of multiple sclerosis. To determine whether up-regulation of MMP-3, or stromelysin-1, was a causative factor during the development of demyelination, we have examined the expression of MMP-3 mRNA and protein in brain tissue of a spontaneously demyelinating mouse model overexpressing DM20 (ND4 line) prior to and during the progression of disease. Stromelysin-1, but not other MMP mRNA was elevated approximately 10-fold in transgenic mice between 5 days and 1 month of age, more than 2 months before the onset of disease, and was coordinately expressed with the DM20 transgene. Stromelysin-1 protein levels were also up-regulated as was tissue inhibitor of metalloproteinase-1 (TIMP-1), an in vivo regulator of stromelysin-1 mRNA. When we crossed our ND4 mice with a line of transgenic mice overexpressing TIMP-1 in brain, clinical signs in these mice were attenuated, and the level of stromelysin-1 protein was reduced. Thus, in this transgenic model of demyelinating disease up-regulation of DM20, MMP-3, and TIMP-1 represent important changes in the chemical pathogenesis in brain, which precede the onset of disease.     

Interaction of arsenic with gap junction protein connexin 43 alters gap junctional intercellular communication

Chronic exposure to Arsenic pollution in ground water is one of the largest environmental health disasters in the world. The toxicity of trivalent Arsenicals primarily happens due to its interaction with sulfhydryl groups in proteins. Arsenic binding to the protein can change the conformation of the protein and alter its interactions with other proteins leading to tissue damage. Therefore, much importance has been given to the studies of Arsenic bound proteins, for the purpose of understanding the origins of toxicity and to explore therapeutics. Here we study the dynamic effect of Arsenic on Connexin 43 (Cx43), a protein that forms the gap junctions, whose alteration deeply perturbs the cell-to-cell communication vital for maintaining tissue homeostasis. In silico molecular modelling and in vitro studies comparing Arsenic treated and untreated conditions show distinct results. Gap junction communication is severely disrupted by Arsenic due to reduced availability of unaltered Cx43 in the membrane bound form. In silico and Inductively Coupled Plasma Mass Spectrometry studies revealed the interaction of Arsenic to the Cx43 preferably occurs through surface exposed cysteines, thereby capping the thiol groups that form disulfide bonds in the tertiary structure. This leads to disruption of Cx43 oligomerization, and altered Cx43 is incompetent for transportation to the membrane surface, often forming aggregates primarily localizing in the endoplasmic reticulum. Loss of functional Cx43 on the cell surface have a deleterious effect on cellular homeostasis leading to selective vulnerability to cell death and tissue damage.