scholarly journals Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

2001 ◽  
Vol 358 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Henning B. BOLDT ◽  
Michael T. OVERGAARD ◽  
Lisbeth S. LAURSEN ◽  
Kathrin WEYER ◽  
Lars SOTTRUP-JENSEN ◽  
...  
Keyword(s):  
Plasma Protein ◽  
Active Site ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Protein A ◽  
Residual Activity ◽  
Zinc Binding ◽  
Binding Motif ◽  
Dependent Manner ◽  
Zinc Binding Motif

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae

2020 ◽  
Vol 168 (4) ◽  
pp. 385-392
Author(s):  
Kaichi Hayashi ◽  
Takeaki Ikeuchi ◽  
Ryo Morishita ◽  
Jun Qian ◽  
Kenji Kojima ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Active Site ◽  
Zinc Binding ◽  
Binding Motif ◽  
Hydrolyze Collagen ◽  
Tyrosine Residues ◽  
Active Variant ◽  
Zinc Binding Motif ◽  
Ionizable Residue

Abstract Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

scholarly journals Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

2001 ◽  
Vol 21 (15) ◽  
pp. 4868-4874 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Brian T. Dwyer ◽  
David Y. Takeda ◽  
Anindya Dutta
Keyword(s):  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Cyclin E ◽  
Cyclin A ◽  
Binding Motif ◽  
Binding Studies ◽  
Alanine Scanning Mutagenesis ◽  
Cyclin Dependent Kinases

ABSTRACT Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

scholarly journals Phosphonamidates are the first phosphorus-based zinc binding motif to show inhibition of β-class carbonic anhydrases from bacteria, fungi, and protozoa

2019 ◽  
Vol 35 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Siham A. Alissa ◽  
Hanan A. Alghulikah ◽  
Zeid A. Alothman ◽  
Sameh M. Osman ◽  
Sonia Del Prete ◽  
...  
Keyword(s):  
Zinc Binding ◽  
Binding Motif ◽  
Carbonic Anhydrases ◽  
Zinc Binding Motif

Conformational dynamics and allosteric effect modulated by the unique zinc-binding motif in class IIa HDACs

2019 ◽  
Vol 21 (23) ◽  
pp. 12173-12183 ◽  
Author(s):  
Huawei Liu ◽  
Fan Zhang ◽  
Kai Wang ◽  
Xiaowen Tang ◽  
Ruibo Wu
Keyword(s):  
Histone Deacetylases ◽  
Conformational Dynamics ◽  
Zinc Binding ◽  
Binding Motif ◽  
Allosteric Effect ◽  
Class Iia Hdacs ◽  
Zinc Binding Motif

Class IIa histone deacetylases (HDACs) have been considered as potential targets for the treatment of several diseases.

scholarly journals Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1146 ◽  
Author(s):  
Brown ◽  
del Corsso ◽  
Zoidl ◽  
Donaldson ◽  
Spray ◽  
...  
Keyword(s):  
Protein Transport ◽  
Mutational Analysis ◽  
Binding Motif ◽  
Dependent Manner ◽  
Electrical Synapses ◽  
Binding Motifs ◽  
Connexin 36 ◽  
Dependent Transport ◽  
Carboxy Terminal

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B–microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.

scholarly journals Pregnancy-Associated Plasma Protein A Induces Inflammatory Cytokine Expression by Activating IGF-I/PI3K/Akt Pathways

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Weiping Li ◽  
Hongwei Li ◽  
Li Zhou ◽  
Zijian Wang ◽  
Bing Hua
Keyword(s):  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Plasma Protein ◽  
Inflammatory Cytokine ◽  
Protein A ◽  
Dependent Manner ◽  
Coronary Syndrome ◽  
Raw264.7 Macrophages ◽  
Igf I

Pregnancy-associated plasma protein A (PAPP-A) was previously reported to be an inflammatory biomarker and a prognostic marker of acute coronary syndrome (ACS) and involved in the process of atherosclerosis and plaque rupture. However, the role of PAPP-A in inflammation is poorly understood. In this study, we aimed to investigate the role of PAPP-A in macrophage activation and inflammatory cytokine production. RAW264.7 macrophages were treated with or without PAPP-A. Reverse-transcriptase quantitative real-time PCR (RT-qPCR) and Western blot were performed to detect gene and protein expressions. The concentration of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in culture supernatants was determined by ELISA. Results showed that PAPP-A significantly stimulated the expression of MCP-1, TNF-α, and IL-6 at both transcriptional and translational levels in a dose-dependent and time-dependent manner. The secretion of these inflammatory cytokines by macrophages was also increased after PAPP-A treatment. Moreover, PAPP-A activated the IGF-I/PI3K/Akt signaling pathway in macrophages. The PAPP-A-mediated upregulation of MCP-1, TNF-α, and IL-6 mRNA and protein levels were strongly inhibited by PI3K inhibitors or IGF-IR siRNA, indicating that the upregulation of MCP-1, TNF-α, and IL-6 could involve the IGF-I/PI3K/Akt pathway. Together, this study demonstrates that PAPP-A activates the macrophage signaling pathway (IGF-I/PI3K/Akt), which drives the expression and production of inflammatory cytokines known to contribute to the initiation and progression of ACS. These findings indicate that PAPP-A may play a proinflammatory role in the pathophysiology of ACS and serve as a potential therapeutic target.

scholarly journals Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

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