Autoactivation of human ADAM8: a novel pre-processing step is required for catalytic activity

2009 ◽  
Vol 29 (4) ◽  
pp. 217-228 ◽  
Author(s):  
Troii Hall ◽  
Joseph W. Leone ◽  
Joseph F. Wiese ◽  
David W. Griggs ◽  
Lyle E. Pegg ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
Activation Mechanism ◽  
Catalytic Domains ◽  
Processing Step ◽  
Inflammatory Stimuli ◽  
Epidermal Growth ◽  
A Disintegrin And Metalloproteinase ◽  
Cysteine Switch

Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu158, which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys167). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.

scholarly journals Aortic Aneurysms, Chronic Kidney Disease and Metalloproteinases

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 194
Author(s):  
Michele Andreucci ◽  
Michele Provenzano ◽  
Teresa Faga ◽  
Ashour Michael ◽  
Gemma Patella ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Proteolytic Enzymes ◽  
Aortic Aneurysms ◽  
Matrix Deposition ◽  
Renal Tubular Cells ◽  
Increased Risk ◽  
Inflammatory Stimuli ◽  
A Disintegrin And Metalloproteinase ◽  
Renal Tubular

Metalloproteinases (MPs) are proteolytic enzymes involved in extracellular matrix deposition, regulation of cellular signals of inflammation, proliferation, and apoptosis. Metalloproteinases are classified into three families: Matrix-MPs (MMPs), A-Disintegrin-and-Metalloprotease (ADAMs), and the A-Disintegrin-and-Metalloproteinase-with-Thrombospondin-1-like-Domains (ADAMTS). Previous studies showed that MPs are involved in the development of aortic aneurysms (AA) and, concomitantly, in the onset of chronic kidney disease (CKD). CKD has been, per se, associated with an increased risk for AA. The aim of this review is to examine the pathways that may associate MPs with CKD and AA. Several MMPs, such as MMP-2, -8, -9, and TIMP-1 have been shown to damage the AA wall and to have a toxic effect on renal tubular cells, leading to fibrosis. Similarly, ADAM10 and 17 have been shown to degrade collagen in the AA wall and to worsen kidney function via pro-inflammatory stimuli, the impairment of the Renin-Angiotensin-Aldosterone System, and the degradation of structural proteins. Moreover, MMP-2 and -9 inhibitors reduced aneurysm growth and albuminuria in experimental and human studies. It would be important, in the future, to expand research on MPs from both a prognostic, namely, to refine risk stratification in CKD patients, and a predictive perspective, likely to improve prognosis in response to targeted treatments.

scholarly journals Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joanna L. Fox ◽  
Michelle A. Hughes ◽  
Xin Meng ◽  
Nikola A. Sarnowska ◽  
Ian R. Powley ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Analysis ◽  
Cell Fate ◽  
Catalytic Domain ◽  
Caspase 8 ◽  
Type I ◽  
Signaling Complex ◽  
Catalytic Domains ◽  
Regulated Cell Death ◽  
Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

scholarly journals SELECTED PROTEOLYTIC ACTIVITY BY EXTRACTS OF DERMESTID LARVAE

2011 ◽  
Vol 19 (19) ◽  
pp. 79-83
Author(s):  
Lavinel G. IONESCU
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Specific Activity ◽  
Proteolytic Enzymes ◽  
High Specific Activity ◽  
Water Extract ◽  
Dermestes Maculatus ◽  
Crude Preparation

The larvae of the Beetle Dermestes maculatus De Geer can subsist on a diet consisting largely of protein. Studies have been undertaken to investigate the nature of proteolytic enzymes. A water extract of the larvae yielded a crude preparation that hydrolyzes gelatin, bide powder, hemoglobin substrate, benzoyl-DL-arginine p-nitroamilide, and glutaryl-L-phenylalanine p-nitroanilide. Enzyme activity was found in a non-dialyzable, heat- and acid0labile portion of the extract yielded two fractions with high specific activity towards gelatin. These are precipitated between 40% to 60% saturation of ammonium sulfate and 60% to 80% saturation. The higher specific activity was observed in the 40%-60% fraction. These results suggest that the larvae of these dermestids contain proteolytic enzymes with actions similar to mammalian trypsin and chymotrypsin. The results also suggest that other proteolytic enzymes may be present as well.

scholarly journals Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. ex4484_79

2021 ◽  
Vol 12 ◽  
Author(s):  
Anke Chen ◽  
Dan Wang ◽  
Rui Ji ◽  
Jixi Li ◽  
Shaohua Gu ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Maximum Activity ◽  
Homologous Proteins ◽  
Glycosidic Bonds ◽  
Catalytic Sites ◽  
Potential Applications ◽  
Beta Glucosidase ◽  
Hydrolysis Of

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

scholarly journals The RpfC (Rv1884) atomic structure shows high structural conservation within the resuscitation-promoting factor catalytic domain

2014 ◽  
Vol 70 (8) ◽  
pp. 1022-1026 ◽  
Author(s):  
Francois-Xavier Chauviac ◽  
Giles Robertson ◽  
Doris H. X. Quay ◽  
Claire Bagnéris ◽  
Christian Dumas ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Catalytic Domain ◽  
Anomalous Scattering ◽  
Molecular Replacement ◽  
Catalytic Domains ◽  
Domain Composition ◽  
Resuscitation Promoting Factors ◽  
Structural Conservation ◽  
High Degree ◽  
Very High

The first structure of the catalytic domain of RpfC (Rv1884), one of the resuscitation-promoting factors (RPFs) fromMycobacterium tuberculosis, is reported. The structure was solved using molecular replacement once the space group had been correctly identified as twinnedP21rather than the apparentC2221by searching for anomalous scattering sites inP1. The structure displays a very high degree of structural conservation with the previously published structures of the catalytic domains of RpfB (Rv1009) and RpfE (Rv2450). This structural conservation highlights the importance of the versatile domain composition of the RPF family.

scholarly journals A Zebrafish Model of Myelodysplastic Syndrome Produced throughtet2Genomic Editing

2014 ◽  
Vol 35 (5) ◽  
pp. 789-804 ◽  
Author(s):  
Evisa Gjini ◽  
Marc R. Mansour ◽  
Jeffry D. Sander ◽  
Nadine Moritz ◽  
Ashley T. Nguyen ◽  
...  
Keyword(s):  
Myelodysplastic Syndrome ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Cpg Islands ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations ofTET2are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain.tet2m/m(homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultanttet2m/mmutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells.tet2m/mzebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity againsttet2mutant cells.

scholarly journals Structural insights into the activation mechanism of dynamin-like EHD ATPases

2017 ◽  
Vol 114 (22) ◽  
pp. 5629-5634 ◽  
Author(s):  
Arthur Alves Melo ◽  
Balachandra G. Hegde ◽  
Claudio Shah ◽  
Elin Larsson ◽  
J. Mario Isas ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Cellular Membrane ◽  
Activation Mechanism ◽  
Gtpase Domain ◽  
Membrane Recruitment ◽  
Spin Resonance ◽  
Epidermal Growth ◽  
Electron Paramagnetic

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

scholarly journals Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate

1991 ◽  
Vol 275 (2) ◽  
pp. 349-353 ◽  
Author(s):  
H C Hawkins ◽  
E C Blackburn ◽  
R B Freedman
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Specific Activity ◽  
Disulphide Bond ◽  
Protein Disulphide Isomerase ◽  
Protein Substrate ◽  
Catalytic Domains ◽  
Standard Assay

1. The activities of protein disulphide-isomerase (PDI) and thioredoxin in catalysing disulphide bond isomerization in a protein substrate were compared by using the standard assay, namely the re-activation of ‘scrambled’ RNAase. 2. The specific activity of PDI was 25-fold greater than that of thioredoxin. 3. The greater efficiency of PDI compared with thioredoxin is considered to be due more to the presence of multiple catalytic domains in PDI than to differences in their active-site sequences. 4. Data and procedures were defined for expressing enzyme activity in standard units, i.e. mumol of active RNAase generated/min.

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