Do synapsin I and microtubule-associated proteins bind to a common site on polymerized tubulin?

International audienceSynapsin I plays an important role in the regulation of neurotransmitter release, since it binds to synaptic vesicles and to the cytoskeleton, and it bundles F-actin and microtubules. We have previously shown by tryptic digestion of synapsin I that a 44 kDa fragment contains a binding site for polymerized tubulin. In the present experiments, we test whether synapsin I and microtubule-associated proteins (MAPs) have the same or a different binding site on tubulin molecules. Our results show that heat stable MAPs do not compete with synapsin I for binding to taxol tubulin. In addition, subtilisin digestion of tubulin, which suppresses MAPs binding, does not abolish synapsin I cosedimentation with taxol tubulin. Thus, our results strongly suggest that synapsin I (as reported for kinesin) does not bind to the 4 kDa subtilisin digested C-terminal part of the tubulin molecule

Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme.

International audienceIsolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme. Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either absorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided.Isolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme. Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either absorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided

Rabbit heart mitochondrial hexokinase: solubilization and general properties.

International audienceIn rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent Km for glucose (0.065 mM), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent Km for ATPMg than the solubilized enzyme, whereas the apparent Km for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968) Proc. Natl. Acad. Sci. USA 61, 598-605]. Furthermore, low concentrations (0.1 mM) of glucose 6-phosphate (G6P) or ATP4- specifically solubilize hexokinase. The solubilizing effect of G6P and ATP4-, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with Pi or Mg2+. In addition, enzyme solubilization by G6P can be reversed by Mg2+ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J. 37, 18-19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent Km for glucose (0.065 mM), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent Km for ATPMg than the solubilized enzyme, whereas the apparent Km for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968) Proc. Natl. Acad. Sci. USA 61, 598-605]. Furthermore, low concentrations (0.1 mM) of glucose 6-phosphate (G6P) or ATP4- specifically solubilize hexokinase. The solubilizing effect of G6P and ATP4-, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with Pi or Mg2+. In addition, enzyme solubilization by G6P can be reversed by Mg2+ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J. 37, 18-19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one

Collagenous sequence governs the trimeric assembly of collagen XII.

International audienceA minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII

Processing in the C-terminal domain of minicollagen XII removes a heparin-binding site.

International audienceA minicollagen comprising the two C-terminal domains of collagen XII (COL1 and NC1) has been expressed in insect cells and characterized biochemically. An interaction with heparin is demonstrated, which depends on the correct folding of the molecule. After secretion, minicollagen XII is immediately processed to a form lacking heparin binding ability. Processed and unprocessed trimers differ only at the level of the eight or nine C-terminal residues but they reveal different structures as judged from rotary shadowing images. Similar processing is also observed in the medium of transfected human HeLa cells. These data show that a heparin-binding site is present in the C-terminal end of the chicken collagen XII sequence and strongly suggest that proteolytic processing in the NC1 domain can occur in vivo and regulate the interactive properties of collagen XII.A minicollagen comprising the two C-terminal domains of collagen XII (COL1 and NC1) has been expressed in insect cells and characterized biochemically. An interaction with heparin is demonstrated, which depends on the correct folding of the molecule. After secretion, minicollagen XII is immediately processed to a form lacking heparin binding ability. Processed and unprocessed trimers differ only at the level of the eight or nine C-terminal residues but they reveal different structures as judged from rotary shadowing images. Similar processing is also observed in the medium of transfected human HeLa cells. These data show that a heparin-binding site is present in the C-terminal end of the chicken collagen XII sequence and strongly suggest that proteolytic processing in the NC1 domain can occur in vivo and regulate the interactive properties of collagen XII

Binding of collagen XIV with the dermatan sulfate side chain of decorin.

International audienceAs an approach to elucidate the role of collagen XIV, which is still unclear, molecules exhibiting affinity for this collagen have been sought in connective tissue. Extracts from fetal bovine tendon were resolved by gel electrophoresis and electrophoretically transferred to nitrocellulose. The blot was overlaid with native collagen XIV and the collagen XIV-binding molecules revealed by immunodecoration with a monoclonal antitype XIV collagen antibody. This experimental approach allowed us to reveal in tendon extracts a diffuse band, with an apparent molecular mass of approximately 100 kDa, that binds collagen XIV. This molecule was also found associated with the fractions containing partially purified type XIV collagen. This 100-kDa molecule was sensitive to chondroitinase ABC and, after chondroitinase digestion, yielded a core protein of about 48 kDa. N-terminal sequence analysis of the proteoglycan after blotting allowed us to identify it as decorin. By solid phase assays we have studied this newly described association between decorin and type XIV collagen and shown that it is a saturable process. In addition, preliminary determination of the domains of the two molecules involved in the association has been performed. The possible role of these interactions is discussed.As an approach to elucidate the role of collagen XIV, which is still unclear, molecules exhibiting affinity for this collagen have been sought in connective tissue. Extracts from fetal bovine tendon were resolved by gel electrophoresis and electrophoretically transferred to nitrocellulose. The blot was overlaid with native collagen XIV and the collagen XIV-binding molecules revealed by immunodecoration with a monoclonal antitype XIV collagen antibody. This experimental approach allowed us to reveal in tendon extracts a diffuse band, with an apparent molecular mass of approximately 100 kDa, that binds collagen XIV. This molecule was also found associated with the fractions containing partially purified type XIV collagen. This 100-kDa molecule was sensitive to chondroitinase ABC and, after chondroitinase digestion, yielded a core protein of about 48 kDa. N-terminal sequence analysis of the proteoglycan after blotting allowed us to identify it as decorin. By solid phase assays we have studied this newly described association between decorin and type XIV collagen and shown that it is a saturable process. In addition, preliminary determination of the domains of the two molecules involved in the association has been performed. The possible role of these interactions is discussed

Degradation of the COL1 domain of type XIV collagen by 92-kDa gelatinase.

International audienceType XIV collagen is a newly described member of the fibril-associated collagens with interrupted triple helices (FACITs). Expression of this collagen has been localized to various embryonic tissues, suggesting that it has a functional role in development. All FACITs thus far described (types IX, XII, XIV, and XVI) contain a highly homologous carboxyl-terminal triple helical domain designated COL1. We have studied the capacity of various matrix metalloproteinases (interstitial collagenase, stromelysin, matrilysin, and 92-kDa gelatinase) to degrade the COL1 domain of collagen XIV. We found that only 92-kDa gelatinase cleaves COL1. Furthermore, digestion of whole native collagen XIV by the 92-kDa gelatinase indicates that this enzyme specifically attacks the carboxyl-terminal triple helix-containing region of the molecule. COL1 is cleaved by 92-kDa gelatinase at 30 degrees C, a full 5-6 degrees C below the melting temperature (Tm) of this domain; native collagen XIV is also degraded at 30 degrees C. In comparison to interstitial collagenase degradation of its physiologic native type I collagen substrate, the 92-kDa enzyme cleaved COL1 (XIV) with comparable catalytic efficacy. Interestingly, following thermal denaturation of the COL1 fragment, its susceptibility to 92-kDa gelatinase increases, but only to a degree that leaves it several orders of magnitude less sensitive to degradation than denatured collagens I and III. These data indicate that native COL1 and collagen XIV are readily and specifically cleaved by 92-kDa gelatinase. They also suggest a role for 92-kDa gelatinase activity in the structural tissue remodeling of the developing embryo.Type XIV collagen is a newly described member of the fibril-associated collagens with interrupted triple helices (FACITs). Expression of this collagen has been localized to various embryonic tissues, suggesting that it has a functional role in development. All FACITs thus far described (types IX, XII, XIV, and XVI) contain a highly homologous carboxyl-terminal triple helical domain designated COL1. We have studied the capacity of various matrix metalloproteinases (interstitial collagenase, stromelysin, matrilysin, and 92-kDa gelatinase) to degrade the COL1 domain of collagen XIV. We found that only 92-kDa gelatinase cleaves COL1. Furthermore, digestion of whole native collagen XIV by the 92-kDa gelatinase indicates that this enzyme specifically attacks the carboxyl-terminal triple helix-containing region of the molecule. COL1 is cleaved by 92-kDa gelatinase at 30 degrees C, a full 5-6 degrees C below the melting temperature (Tm) of this domain; native collagen XIV is also degraded at 30 degrees C. In comparison to interstitial collagenase degradation of its physiologic native type I collagen substrate, the 92-kDa enzyme cleaved COL1 (XIV) with comparable catalytic efficacy. Interestingly, following thermal denaturation of the COL1 fragment, its susceptibility to 92-kDa gelatinase increases, but only to a degree that leaves it several orders of magnitude less sensitive to degradation than denatured collagens I and III. These data indicate that native COL1 and collagen XIV are readily and specifically cleaved by 92-kDa gelatinase. They also suggest a role for 92-kDa gelatinase activity in the structural tissue remodeling of the developing embryo

Alternative splicing of type II procollagen pre-mRNA in chondrocytes is oppositely regulated by BMP-2 and TGF-beta1.

International audienceType II collagen is the major protein of cartilage and is synthesized as a procollagen in two forms (IIA and IIB), generated by differential splicing of the gene primary transcript. Previous studies have indicated that only type IIB is expressed in differentiated chondrocytes. Here, we examined the effects of bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 on the expression of IIA and IIB forms expressed in de-differentiated chondrocytes grown in monolayer. Our results demonstrate that BMP-2 favors expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These observations reveal the specific capability of BMP-2 to reverse the de-differentiation state of chondrocytes.Type II collagen is the major protein of cartilage and is synthesized as a procollagen in two forms (IIA and IIB), generated by differential splicing of the gene primary transcript. Previous studies have indicated that only type IIB is expressed in differentiated chondrocytes. Here, we examined the effects of bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 on the expression of IIA and IIB forms expressed in de-differentiated chondrocytes grown in monolayer. Our results demonstrate that BMP-2 favors expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These observations reveal the specific capability of BMP-2 to reverse the de-differentiation state of chondrocytes