scholarly journals Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery

Proteomes ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 36 ◽  
Author(s):  
Raj Luxmi ◽  
Crysten Blaby-Haas ◽  
Dhivya Kumar ◽  
Navin Rauniyar ◽  
Stephen M. King ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Sexual Reproduction ◽  
Growth Factor Signaling ◽  
Carboxypeptidase B ◽  
Peptide Processing ◽  
Processing Enzymes ◽  
Unicellular Green Alga ◽  
Sds Page ◽  
Catalytically Active ◽  
Cysteine Endopeptidases

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

scholarly journals Proteolytic Events of Wound-Healing — Coordinated Interactions Among Matrix Metalloproteinases (MMPs), Integrins, and Extracellular Matrix Molecules

2001 ◽  
Vol 12 (5) ◽  
pp. 373-398 ◽  
Author(s):  
Bjorn Steffensen ◽  
Lari Häkkinen ◽  
Hannu Larjava
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Matrix Metalloproteinases ◽  
Wound Repair ◽  
Enzyme Activation ◽  
Processing Enzymes ◽  
The Matrix ◽  
Signaling Receptors ◽  
State Of Rest ◽  
And Function

During wound-healing, cells are required to migrate rapidly into the wound site via a proteolytically generated pathway in the provisional matrix, to produce new extracellular matrix, and, subsequently, to remodel the newly formed tissue matrix during the maturation phase. Two classes of molecules cooperate closely to achieve this goal, namely, the matrix adhesion and signaling receptors, the integrins, and matrix-degrading and -processing enzymes, the matrix metalloproteinases (MMPs). There is now substantial experimental evidence that blocking key molecules of either group will prevent or seriously delay wound-healing. It has been known for some time now that cell adhesion by means of the integrins regulates the expression of MMPs. In addition, certain MMPs can bind to integrins or other receptors on the cell surface involved in enzyme activation, thereby providing a mechanism for localized matrix degradation. By proteolytically modifying the existing matrix molecules, the MMPs can then induce changes in cell behavior and function from a state of rest to migration. During wound repair, the expression of integrins and MMPs is simultaneously up-regulated. This review will focus on those aspects of the extensive knowledge of fibroblast and keratinocyte MMPs and integrins in biological processes that relate to wound-healing.

Key peptide processing enzymes are expressed by breast cancer cells

2001 ◽  
Vol 165 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Jinlin Du ◽  
Brendan P. Keegan ◽  
William G. North
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Peptide Processing ◽  
Processing Enzymes

Purification and Characterization of Cytochrome c6 from the Unicellular Green Alga Scenedesmus obliquus

1997 ◽  
Vol 52 (11-12) ◽  
pp. 740-746 ◽  
Author(s):  
Röbbe Wünschiers ◽  
Thomas Zinn ◽  
Dietmar Linder ◽  
Rüdiger Schulz
Keyword(s):  
Cytochrome C ◽  
Green Alga ◽  
Scenedesmus Obliquus ◽  
Terminal Sequence ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Unicellular Green Alga ◽  
Sds Page ◽  
Monoraphidium Braunii

Abstract Purification of a soluble cytochrome c6 from the unicellular green alga Scenedesmus obliquus by a simple and rapid method is described. The purification procedure includes ammonium sulfate precipitation and non-denaturating PAGE. The N-terminal sequence of the first 20 amino acids was determined and shows 85% similarity and 75% identity to the sequence of cytochrome c6 from the green alga Monoraphidium braunii. The ferrocyto-chrome shows typical UV/VIS absorption peaks at 552.9, 521.9 and 415.7 nm. The apparent molecular mass was estimated to be 12 kD a by SDS-PAGE. EPR-spectroscopy at 20K shows resonances indicative for two distinct low-spin heme forms.

scholarly journals In Vivo Dimerization of Types 1, 2, and 3 Iodothyronine Selenodeiodinases

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 937-946 ◽  
Author(s):  
Cyntia Curcio-Morelli ◽  
Balazs Gereben ◽  
Ann Marie Zavacki ◽  
Brian W. Kim ◽  
Stephen Huang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Western Blot ◽  
Relative Molecular Mass ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Cell Lysate ◽  
Sds Page ◽  
Catalytically Active

The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of 75Se-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2–5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (Mr) of 55,000 for D1, Mr 62,000 for D2, and Mr 65,000 for D3, which were eliminated by 100 mm dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that overexpressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.

scholarly journals Activation of a phospholipase Cβ2 deletion mutant by limited proteolysis

1998 ◽  
Vol 330 (1) ◽  
pp. 461-468 ◽  
Author(s):  
Petra SCHNABEL ◽  
Montserrat CAMPS
Keyword(s):  
Deletion Mutant ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Cell System ◽  
Heterotrimeric G Proteins ◽  
Sds Page ◽  
Catalytically Active ◽  
Heterodimeric Complex ◽  
High Degree ◽  
Hydrolysis Of

All phosphoinositide-specific phospholipases C (PLC) identified until today exhibit a high degree of similarity within two regions of 170 and 260 residues, respectively, which are designated regions X and Y. The PLCβ family, including four members designated PLCβ1, PLCβ2, PLCβ3 and PLCβ4, is regulated by heterotrimeric G proteins. In order to investigate structure-function relationships of PLCβ2, we expressed PLCβ2Δ, a deletion mutant of PLCβ2 which lacks most of the sequence downstream of region Y, in the baculovirus/insect cell system. The mutant was present in both soluble and particulate fractions of Sf9 cells and was demonstrated to be catalytically active and sensitive to βγ-subunits. Sf9 cytosol containing this mutant was subjected to limited proteolysis by trypsin and S. aureus protease V8, respectively. Immunochemical analysis revealed that both proteases cleaved the enzyme between the regions X and Y. Most interestingly, proteolytic cleavage at this site by both proteases stimulated the catalytic activity of PLC2β2Δ. The proteolytically activated enzyme was still sensitive to βγ-subunits and showed an unchanged concentration dependence on Ca2+. Gel filtration chromatography indicated that the fragments generated by cleavage between the regions X and Y were still connected and formed a functional heterodimeric complex. In order to visualize all fragments generated by protease V8, PLCβ2Δ was purified to homogeneity from Sf9 cytosol. Limited proteolysis of the purified enzyme by S. aureus protease V8 and subsequent SDS/PAGE and silver staining revealed that several cuts take place between the regions X and Y and that the resulting fragments remain intact. We hypothesize that the activating proteolytic cut induces a conformational change of the enzyme which might facilitate hydrolysis of the phospholipid substrate.

Reaction mechanism of matrix metalloproteinases with a catalytically active zinc ion studied by the QM(DFTB)/MM simulations

2016 ◽  
Vol 26 (3) ◽  
pp. 209-211 ◽  
Author(s):  
Tatiana Vasilevskaya ◽  
Maria G. Khrenova ◽  
Alexander V. Nemukhin ◽  
Walter Thiel
Keyword(s):  
Reaction Mechanism ◽  
Matrix Metalloproteinases ◽  
Zinc Ion ◽  
Catalytically Active

scholarly journals Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung

1998 ◽  
Vol 331 (1) ◽  
pp. 69-78 ◽  
Author(s):  
José M. LIZCANO ◽  
Keith F. TIPTON ◽  
Mercedes UNZETA
Keyword(s):  
Amine Oxidase ◽  
Gel Filtration ◽  
Integral Membrane Protein ◽  
Significant Activity ◽  
Bovine Plasma ◽  
Sds Page ◽  
Catalytically Active ◽  
Cibacron Blue 3Ga ◽  
Membrane Bound ◽  
Triton X

Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bovine lung microsomes in a form which is catalytically active and stable to storage. The enzyme, an integral membrane protein, was solubilized with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxylapatite, Lens culinaris-agarose, Resource Q-FPLC and gel filtration on Superdex 200 HR-FPLC. This is the first reported procedure for the extensive purification of a membrane-bound SSAO. The purified enzyme had an apparent Mr of 400000 but exhibited microheterogeneity with SDS/PAGE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent Mr values of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling staining and reaction with carbonyl reagents were consistent with the presence of a quinone cofactor, possibly topa quinone. The enzyme was also shown to contain two mol of Cu/mol of enzyme and removal of half of this bound copper resulted essentially in complete inhibition of enzyme activity. In contrast to the reported behaviour of the SSAO enzymes from plasma, the bovine lung enzyme was relatively insensitive to inhibition by cyanide, copper-chelating agents and amiloride. The specificity of the bovine lung enzyme was also narrower than reported for soluble SSAO. It catalysed the oxidative deamination of benzylamine, methylamine, 2-phenylethylamine and histamine but had no significant activity towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine.

scholarly journals Asparagine-linked glycoprotein biosynthesis in rat epididymis. Presence of a mannosidase II-like enzyme

1991 ◽  
Vol 277 (1) ◽  
pp. 213-221 ◽  
Author(s):  
M D Skudlarek ◽  
M C Orgebin-Crist ◽  
D R P Tulsiani
Keyword(s):  
Antigenic Site ◽  
Indirect Evidence ◽  
Similar Function ◽  
Membrane Component ◽  
Complex Type ◽  
Present Evidence ◽  
Processing Enzymes ◽  
Golgi Membranes ◽  
Sds Page ◽  
Rat Epididymis

Previous studies from this laboratory using p-nitrophenyl alpha-D-mannoside (p-NPM) as substrate provided no evidence for the presence of mannosidase II in the rat epididymis [Skudlarek & Orgebin-Crist (1988) J. Reprod. Fertil. 84, 611-617]. However, rat epididymal epithelial cells cultured in the presence of swainsonine, an inhibitor of mannosidase II, produce abnormally processed N-linked glycoproteins containing hybrid-type oligosaccharides instead of complex-type [Tulsiani, Skudlarek & Orgebin-Crist (1990) Biol. Reprod. 43, 130-138], a result providing indirect evidence for the presence of mannosidase II-like enzyme in rat epididymis. In the studies described here, we present evidence for the occurrence of this processing enzyme in rat epididymal Golgi membranes. This enzyme is an integral Golgi membrane component. Like liver mannosidase II, the epididymal enzyme cleaves alpha 1,3- and alpha 1,6-linked mannosyl residues from GlcNAcMan5GlcNAc. However, unlike liver mannosidase II, the epididymal enzyme shows no activity towards the synthetic substrate, p-NPM. The epididymal mannosidase cross-reacts with liver anti-(mannosidase II) antibody, a result suggesting that the two enzymes share a common antigenic site(s). Immunoblotting studies following resolution of liver and epididymal Golgi membranes on SDS/PAGE show that, whereas the liver mannosidase II was resolved as a doublet of Mr 120,000 and 122,000, only the Mr 120,000 band was observed in the epididymal Golgi membranes. Immunoblotting of the Golgi-rich fractions, resolved under non-denaturing conditions, showed different patterns of charge and/or size isomers from the two tissues. These studies demonstrate tissue-specific differences in processing enzymes with similar function.

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