The Inactivation of a New Peptidoglycan Hydrolase Pmp23 Leads to Abnormal Septum Formation in Streptococcus pneumoniae

The bacterial peptidoglycan is the major component of the cell wall which integrity is essential to cell survival. In a previous work, we identified, in the positive-Gram pathogen Streptococcus pneumoniae , a unique protein containing a new putative peptidoglycan hydrolytic domain named PECACE (PEptidoglycan CArbohydrate Cleavage Enzyme). In this study, we characterise the physiological function of this protein called Pmp23 (Pneumococcal Membrane Protein of 23 kDa). A cell wall hydrolytic activity is observed with the recombinant protein. Inactivation of the pmp23 gene in the pneumococcus led to a decreased flocculation, an increased sensitivity to β-lactam antibiotics and morphological alterations affecting the formation and localisation of the division septa. Taken together these observations indicate that Pmp23 is a hydrolase whose function is linked to peptidoglycan metabolism at the septum site

Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum.

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230]

Mechanisms of collagen trimer assembly

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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