scholarly journals Structure/Function Relationships in the Minicollagen ofHydraNematocysts

2002 ◽  
Vol 277 (51) ◽  
pp. 49200-49204 ◽  
Author(s):  
Suat Özbek ◽  
Olivier Pertz ◽  
Martine Schwager ◽  
Ariel Lustig ◽  
Thomas Holstein ◽  
...  
Keyword(s):  
Triple Helix ◽  
Molecular Structures ◽  
Disulfide Linkage ◽  
Collagen Triple Helix ◽  
Rich Domain ◽  
Reducing Conditions ◽  
Cross Links ◽  
Polymeric Structures ◽  
Cysteine Rich Domain

The minicollagens found in the inner layer of theHydranematocyst walls are the smallest collagens known with 12–16 Gly-X-Yrepeats. Minicollagen-1, the best characterized member of this protein family so far, consists of a central collagen triple helix of 12 nm in length flanked at both ends by a polyproline stretch and a conserved cysteine-rich domain. The cysteine-rich tails are proposed to function in the assembly of soluble minicollagen trimers to high molecular structures by a switch of the disulfide linkage from intramolecular to intermolecular bonds. In this study, we investigate the trimeric nature of minicollagen-1 and its capacity to form disulfide-linked polymersin vitro. A fusion protein of minicollagen-1 with maltose-binding protein is secreted as a soluble trimer with only intrachain and no interchain disulfide bridges as confirmed by melting the collagen triple helix under reducing and non-reducing conditions. The conversion of minicollagen-1 trimers to monomers takes place between 40 and 55 °C with the melting point being ∼45 °C. Oxidative reshuffling of the minicollagen-1 trimers leads to the formation of high molecular aggregates, which upon reduction show distinct polytrimeric states. Minicollagen trimers in isolated nematocyst capsules proved to be sensitive to SDS and were engaged in polymeric structures with additional cross-links that were resistant to reducing agent.

scholarly journals Isolation of a novel bone glycosylated phosphoprotein with disulphide cross-links to osteonectin

1998 ◽  
Vol 330 (3) ◽  
pp. 1423-1431 ◽  
Author(s):  
Hai-Yan ZHOU ◽  
Erdjan SALIH ◽  
J. Melvin GLIMCHER
Keyword(s):  
Molecular Forms ◽  
Experimental Conditions ◽  
Post Translational Modifications ◽  
Disulphide Bonds ◽  
Homology Searching ◽  
Reducing Conditions ◽  
Neutral Sugars ◽  
Cross Links

An 80 kDa protein was purified from calf bone by HCl-demineralization followed by 0.5 M EDTA/1.0 M NaCl extraction and sequential chromatography on DE-52, hydroxyapatite, and TSK-gel G3000SW HPLC columns. From the DE-52 column the protein was eluted at three different fractions, of which one further separated into two fractions on the hydroxyapatite column, indicating that the protein is present in four different molecular forms designated as 80 k-I-1, k-I-2, k-II, k-III. The N-terminal sequence analysis of all four forms gave the same sequence, SEQYNQEPNNV. Several tryptic internal peptides were also generated, purified and sequenced, leading to the identification of several repeat sequences, IFLGXXEI. Homology searching of the N-terminal and internal sequences indicates that this is a novel protein. Both 80 k-I-2 and k-III had similar amino acid composition with high contents of Asx, Glx and Leu and contained 7 and 16 phosphoserines per 1000 total amino acids, respectively. The 80 k-I-1 and 80 k-II forms were stained with Rhodamine B specific for phosphoproteins. The four forms contained different contents of neutral sugars ranging from 5.5 to 26% (w/w protein) and ~ 1.7% sialic acid. These data indicated that the 80 kDa protein exists in four isomeric forms, at least based on the different post-translational modifications. The evaluation of the 80 kDa glycosylated phosphoprotein under alkylating, reducing and non-reducing conditions indicated that this protein undergoes polymerization through intermolecular disulphide bonds. Furthermore, the 80 kDa protein and osteonectin (ON), both of which are cysteine-rich proteins, can cross-link with each other via disulphide bonds, and this process can be induced to take place in vitro under experimental conditions. The occurrence of such a phenomenon in vivo was confirmed from the presence of similar high Mr components containing both 80 kDa and ON in the same SDS/PAGE bands, detected by the respective antibody reactions in crude bone extracts which were extracted in the presence of alkylating agent.

scholarly journals The conserved cysteine-rich domain of a tesmin/TSO1-like protein binds zinc in vitro and TSO1 is required for both male and female fertility in Arabidopsis thaliana

2007 ◽  
Vol 58 (13) ◽  
pp. 3657-3670 ◽  
Author(s):  
Stig Uggerhøj Andersen ◽  
Randi Groslier Algreen-Petersen ◽  
Martina Hoedl ◽  
Anna Jurkiewicz ◽  
Cristina Cvitanich ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Female Fertility ◽  
Male And Female ◽  
Rich Domain ◽  
Cysteine Rich Domain

scholarly journals Increased Susceptibility of  Thymocytes to Apoptosis in Mice Lacking AIM, a Novel Murine Macrophage-derived Soluble Factor Belonging to the Scavenger Receptor Cysteine-rich Domain Superfamily

1999 ◽  
Vol 189 (2) ◽  
pp. 413-422 ◽  
Author(s):  
Toru Miyazaki ◽  
Yumiko Hirokami ◽  
Nobuyuki Matsuhashi ◽  
Hisakazu Takatsuka ◽  
Makoto Naito
Keyword(s):  
Scavenger Receptor ◽  
The Body ◽  
Murine Macrophage ◽  
Double Positive ◽  
Differential Signal ◽  
Rich Domain ◽  
Macrophage Scavenger Receptor ◽  
Cysteine Rich Domain

Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection.

Collagen Triple Helix Repeat Containing 1 Promotes Melanoma Cell Adhesion and Survival

2011 ◽  
Vol 15 (2) ◽  
pp. 103-110 ◽  
Author(s):  
Wency Ip ◽  
Olivier Wellman-Labadie ◽  
Liren Tang ◽  
Mingwan Su ◽  
Richard Yu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Triple Helix ◽  
Potential Method ◽  
Melanoma Cells ◽  
Conventional Chemotherapy ◽  
Cellular Processes ◽  
Collagen Triple Helix ◽  
Cell Changes ◽  
Attachment Ability

Background: The extracellular protein collagen triple helix repeat containing 1 (CTHRC1) is aberrantly upregulated in melanoma and most human solid cancers. However, its role in cancer remains unknown. Objective: In this study, we investigated the functional impact of CTHRC1 on melanoma cells in vitro. Methods: Stable clones of cultured melanoma cells expressing different amounts of CTHRC1 protein were generated and evaluated to characterize their growth, survival, and attachment ability as well as their sensitivity to chemotherapy. Results: In cultured MMAN and MMRU melanoma cells, increased expression of CTHRC1 protein resulted in morphologic cell changes, enhanced cell adhesion to culture surfaces, increased cell proliferation, and decreased apoptosis. Furthermore, decreased CTHRC1 expression through antisense inhibition enhanced temozolomide sensitivity. Conclusion: CTHRC1 expression influences cellular processes, including cell adhesion and survival. Additionally, CTHRC1 inhibition may represent a potential method for decreasing melanoma resistance to conventional chemotherapy.

scholarly journals Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo

2003 ◽  
Vol 163 (5) ◽  
pp. 1045-1055 ◽  
Author(s):  
Patricia A. Loomis ◽  
Lili Zheng ◽  
Gabriella Sekerková ◽  
Benjarat Changyaleket ◽  
Enrico Mugnaini ◽  
...  
Keyword(s):  
Hair Cells ◽  
Actin Polymerization ◽  
Cytochalasin D ◽  
Actin Monomer ◽  
Actin Bundles ◽  
Rich Domain ◽  
Cross Links ◽  
Necessary And Sufficient

The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at ∼1.5 s−1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.

scholarly journals Mini-collagens in hydra nematocytes.

1991 ◽  
Vol 115 (4) ◽  
pp. 1159-1169 ◽  
Author(s):  
E M Kurz ◽  
T W Holstein ◽  
B M Petri ◽  
J Engel ◽  
C N David
Keyword(s):  
Amino Acid ◽  
Central Region ◽  
Triple Helix ◽  
Amino Acid Sequences ◽  
Disulfide Linkage ◽  
Amino Terminal ◽  
Collagen Triple Helix ◽  
Polyproline Ii ◽  
Carboxy Terminal

We have isolated and characterized four collagen-related c-DNA clones (N-COL 1, N-COL 2, N-COL 3, N-COL 4) that are highly expressed in developing nematocytes in hydra. All four c-DNAs as well as their corresponding transcripts are small in size (600-1,000 bp). The deduced amino acid sequences show that they contain a central region consisting of 14 to 16 Gly-X-Y triplets. This region is flanked amino-terminal by a stretch of 14-23 proline residues and carboxy-terminal by a stretch of 6-9 prolines. At the NH2- and COOH-termini are repeated patterns of cysteine residues that are highly conserved between the molecules. A model is proposed which consists of a central stable collagen triple helix of 12-14 nm length from which three 9-22 nm long polyproline II type helices emerge at both ends. Disulfide linkage between cysteine-rich segments in these helices could lead to the formation of oligomeric network structures. Electrophoretic characterization of nematocyst extracts allows resolution of small proline-rich polypeptides that correspond in size to the cloned sequences.

scholarly journals The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

2005 ◽  
Vol 170 (7) ◽  
pp. 1091-1099 ◽  
Author(s):  
Jessica E. Smotrys ◽  
Marissa J. Schoenfish ◽  
Monica A. Stutz ◽  
Maurine E. Linder
Keyword(s):  
Membrane Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Vacuolar Membrane ◽  
Membrane Association ◽  
Rich Domain ◽  
Vacuole Fusion ◽  
Cysteine Rich Domain

Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145–3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133–35140). Proteins that contain an Asp-His-His-Cys (DHHC)–cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.

scholarly journals Mechanistic Insight into the Binding and Swelling Functions of Prepeptidase C-Terminal (PPC) Domains from Various Bacterial Proteases

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
JiaFeng Huang ◽  
RiBang Wu ◽  
Dan Liu ◽  
BinQiang Liao ◽  
Ming Lei ◽  
...  
Keyword(s):  
Triple Helix ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Systematic Research ◽  
Collagen Binding ◽  
Content Type ◽  
Collagen Triple Helix ◽  
Insoluble Collagen ◽  
Hydrophobic Binding ◽  
Cross Links

ABSTRACT The bacterial prepeptidase C-terminal (PPC) domain can be found in the C termini of a wide variety of proteases that are secreted by marine bacteria. However, the functions of these PPC domains remain unknown due to a lack of systematic research. Here, the binding and swelling abilities of eight PPC domains from six different proteases were compared systematically via scanning electron microscopy (SEM), enzyme assays, and fluorescence spectroscopy. These PPC domains all possess the ability to bind and swell insoluble collagen. PPC domains can expose collagen monomers but cannot disrupt the pyridinoline cross-links or unwind the collagen triple helix. This ability can play a synergistic role alongside collagenase in collagen hydrolysis. Site-directed mutagenesis of the PPC domain from Vibrio anguillarum showed that the conserved polar and aromatic residues Y6, D26, D28, Y30, W42, E53, C55, and Y65 and the hydrophobic residues V10, V18, and I57 played key roles in substrate binding. Molecular dynamic simulations were conducted to investigate the interactions between PPC domains and collagen. Most PPC domains have a similar mechanism for binding collagen, and the hydrophobic binding pocket of PPC domains may play an important role in collagen binding. This study sheds light on the substrate binding mechanisms of PPC domains and reveals a new function for the PPC domains of bacterial proteases in substrate degradation. IMPORTANCE Prepeptidase C-terminal (PPC) domains commonly exist in the C termini of marine bacterial proteases. Reports examining PPC have been limited, and its functions remain unclear. In this study, eight PPCs from six different bacteria were examined. Most of the PPCs possessed the ability to bind collagen, feathers, and chitin, and all PPCs could significantly swell insoluble collagen. PPCs can expose collagen monomers but cannot disrupt pyridinoline cross-links or unwind the collagen triple helix. This swelling ability may also play synergistic roles in collagen hydrolysis. Comparative structural analyses and the examination of PPC mutants revealed that the hydrophobic binding pockets of PPCs may play important roles in collagen binding. This study provides new insights into the functions and ecological significance of PPCs, and the molecular mechanism of the collagen binding of PPCs was clarified, which is beneficial for the protein engineering of highly active PPCs and collagenase in the pharmaceutical industry and of artificial biological materials.

scholarly journals The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase

2002 ◽  
Vol 159 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Amy F. Roth ◽  
Ying Feng ◽  
Linyi Chen ◽  
Nicholas G. Davis
Keyword(s):  
Reaction Mechanism ◽  
Enzymatic Reaction ◽  
Conserved Sequence ◽  
Rich Domain ◽  
Protein Palmitoylation ◽  
Domain Protein ◽  
Polytopic Membrane Protein ◽  
Cysteine Rich Domain

Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.

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