scholarly journals Metal Preferences of Zinc-Binding Motif on Metalloproteases

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Kayoko M. Fukasawa ◽  
Toshiyuki Hata ◽  
Yukio Ono ◽  
Junzo Hirose
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Copper Ion ◽  
Binding Motif ◽  
Wild Type ◽  
Zinc Metalloprotease ◽  
Naturally Occurring ◽  
Zinc Metalloproteases ◽  
Almost All ◽  
Zinc Binding Motif

Almost all naturally occurring metalloproteases are monozinc enzymes. The zinc in any number of zinc metalloproteases has been substituted by some other divalent cation. Almost all Co(II)- or Mn(II)-substituted enzymes maintain the catalytic activity of their zinc counterparts. However, in the case of Cu(II) substitution of zinc proteases, a great number of enzymes are not active, for example, thermolysin, carboxypeptidase A, endopeptidase from Lactococcus lactis, or aminopeptidase B, while some do have catalytic activity, for example, astacin (37%) and DPP III (100%). Based on structural studies of various metal-substituted enzymes, for example, thermolysin, astacin, aminopeptidase B, dipeptidyl peptidase (DPP) III, and del-DPP III, the metal coordination geometries of both active and inactive Cu(II)-substituted enzymes are shown to be the same as those of the wild-type Zn(II) enzymes. Therefore, the enzyme activity of a copper-ion-substituted zinc metalloprotease may depend on the flexibility of catalytic domain.

scholarly journals Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

2005 ◽  
Vol 391 (2) ◽  
pp. 285-289 ◽  
Author(s):  
Nandita S. Raikwar ◽  
Rosario F. Bowen ◽  
Mark A. Deeg
Keyword(s):  
Catalytic Activity ◽  
Phospholipase D ◽  
Catalytic Domain ◽  
Computer Modelling ◽  
Critical Role ◽  
Biochemical Properties ◽  
Catalytic Site ◽  
Wild Type ◽  
Histidine Residues ◽  
Hydrolysis Of

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.

scholarly journals The zinc-binding motif in tankyrases is required for the structural integrity and proper function of the catalytic domain

2021 ◽  
Author(s):  
Sven T Sowa ◽  
Lari Lehtiö
Keyword(s):  
Structural Integrity ◽  
Catalytic Domain ◽  
Zinc Binding ◽  
Structural Defects ◽  
Proper Function ◽  
Binding Motif ◽  
Acceptor Site ◽  
Structural Element ◽  
Zinc Binding Motif

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and they are therefore possible drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyl-transferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on activity, stability and structure of human tankyrases. We generated mutants of human TNKS1 and TNKS2 abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive likely due to introduction of major structural defects, the TNKS2 mutant remained active and displayed a different activity profile compared to the wild type.

scholarly journals The tumour-suppressor function of PTEN requires an N-terminal lipid-binding motif

2004 ◽  
Vol 379 (2) ◽  
pp. 301-307 ◽  
Author(s):  
Steven M. WALKER ◽  
Nick R. LESLIE ◽  
Nevin M. PERERA ◽  
Ian H. BATTY ◽  
C. Peter DOWNES
Keyword(s):  
Catalytic Activity ◽  
Lipid Binding ◽  
Tumour Suppressor ◽  
Binding Motif ◽  
Loss Of Function ◽  
Wild Type ◽  
Suppressor Function ◽  
Tumour Suppressor Function ◽  
Anionic Lipids

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour-suppressor protein is a phosphoinositide 3-phosphatase which antagonizes phosphoinositide 3-kinase-dependent signalling by dephosphorylating PtdIns(3,4,5)P3. Most tumour-derived point mutations of PTEN induce a loss of function, which correlates with profoundly reduced catalytic activity. However, here we characterize a point mutation at the N-terminus of PTEN, K13E from a human glioblastoma, which displayed wild-type activity when assayed in vitro. This mutation occurs within a conserved polybasic motif, a putative PtdIns(4,5)P2-binding site that may participate in membrane targeting of PTEN. We found that catalytic activity against lipid substrates and vesicle binding of wild-type PTEN, but not of PTEN K13E, were greatly stimulated by anionic lipids, especially PtdIns(4,5)P2. The K13E mutation also greatly reduces the efficiency with which anionic lipids inhibit PTEN activity against soluble substrates, supporting the hypothesis that non-catalytic membrane binding orientates the active site to favour lipid substrates. Significantly, in contrast to the wild-type enzyme, PTEN K13E failed either to prevent protein kinase B/Akt phosphorylation, or inhibit cell proliferation when expressed in PTEN-null U87MG cells. The cellular functioning of K13E PTEN was recovered by targeting to the plasma membrane through inclusion of a myristoylation site. Our results establish a requirement for the conserved N-terminal motif of PTEN for correct membrane orientation, cellular activity and tumour-suppressor function.

Zinc Ligands in an Astacin Family Metalloprotease Meprin A

2002 ◽  
Vol 383 (7-8) ◽  
pp. 1167-1173 ◽  
Author(s):  
B. A. Doll ◽  
J. P. Villa ◽  
F. T. Ishmael ◽  
J. S. Bond
Keyword(s):  
Enzymatic Activity ◽  
Zinc Content ◽  
Zinc Binding ◽  
Binding Motif ◽  
Wild Type ◽  
Mutant Proteins ◽  
Substrate Analog ◽  
Sitedirected Mutagenesis ◽  
Extensive Degradation ◽  
Zinc Binding Motif

Abstract A conserved tyrosine residue in the astacin family of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Sitedirected mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin α was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin α protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalentlylinked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wildtype protein. The zinc content in the wildtype and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.

Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association

2000 ◽  
Vol 351 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Michele T. YIP-SCHNEIDER ◽  
Wenyan MIAO ◽  
Amy LIN ◽  
Darlene S. BARNARD ◽  
Guri TZIVION ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Catalytic Domain ◽  
Adaptor Protein ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Regulatory Domain ◽  
Wild Type

The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.

Evaluation of flow-cytometry for the monitoring of infectious human rotavirus in water

1997 ◽  
Vol 35 (11-12) ◽  
pp. 451-453
Author(s):  
F. X. Abad ◽  
A. Bosch ◽  
J. Comas ◽  
D. Villalba ◽  
R. M. Pintó
Keyword(s):  
Flow Cytometry ◽  
Water Samples ◽  
Wild Type ◽  
Cell Monolayers ◽  
Naturally Occurring ◽  
Human Rotavirus ◽  
Faecal Samples

A method has been developed for the detection of infectious human rotavirus (HRV), based on infection of MA104 and CaCo-2 cell monolayers and ulterior flow cytometry. The sensitivity of the flow cytometry procedure for the cell-adapted HRV enabled the detection of 200 and 2 MPNCU in MA104 and CaCo-2 cells, respectively. Flow cytometry performed five days after infection of CaCo-2 enabled the detection of naturally occurring wild-type HRV in faecal samples and concentrated water samples.

scholarly journals The hobo Transposable Element Excises and Has Related Elements in Tephritid Species

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1339-1347
Author(s):  
Alfred M Handler ◽  
Sheilachu P Gomez
Keyword(s):  
Drosophila Melanogaster ◽  
Ceratitis Capitata ◽  
Bactrocera Dorsalis ◽  
Parsimony Analysis ◽  
Wild Type ◽  
Anastrepha Suspensa ◽  
Tephritid Species ◽  
Mutant Strains ◽  
Almost All ◽  
Horizontal Transfers

Abstract Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobe-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.

scholarly journals Effects of B.1.1.7 and B.1.351 on COVID-19 Dynamics: A Campus Reopening Study

2021 ◽  
Author(s):  
Kevin Linka ◽  
Mathias Peirlinck ◽  
Amelie Schäfer ◽  
Oguz Ziya Tikenogullari ◽  
Alain Goriely ◽  
...  
Keyword(s):  
Online Education ◽  
The United States ◽  
Wild Type ◽  
Seir Model ◽  
Population Mixing ◽  
Santa Clara County ◽  
Increased Risk ◽  
Controversial Topic ◽  
Almost All ◽  
Local Reproduction

AbstractThe timing and sequence of safe campus reopening has remained the most controversial topic in higher education since the outbreak of the COVID-19 pandemic. By the end of March 2020, almost all colleges and universities in the United States had transitioned to an all online education and many institutions have not yet fully reopened to date. For a residential campus like Stanford University, the major challenge of reopening is to estimate the number of incoming infectious students at the first day of class. Here we learn the number of incoming infectious students using Bayesian inference and perform a series of retrospective and projective simulations to quantify the risk of campus reopening. We create a physics-based probabilistic model to infer the local reproduction dynamics for each state and adopt a network SEIR model to simulate the return of all undergraduates, broken down by their year of enrollment and state of origin. From these returning student populations, we predict the outbreak dynamics throughout the spring, summer, fall, and winter quarters using the inferred reproduction dynamics of Santa Clara County. We compare three different scenarios: the true outbreak dynamics under the wild-type SARS-CoV-2, and the hypothetical outbreak dynamics under the new COVID-19 variants B.1.1.7 and B.1.351 with 56% and 50% increased transmissibility. Our study reveals that even small changes in transmissibility can have an enormous impact on the overall case numbers. With no additional countermeasures, during the most affected quarter, the fall of 2020, there would have been 203 cases under baseline reproduction, compared to 4727 and 4256 cases for the B.1.1.7 and B.1.351 variants. Our results suggest that population mixing presents an increased risk for local outbreaks, especially with new and more infectious variants emerging across the globe. Tight outbreak control through mandatory quarantine and test-trace-isolate strategies will be critical in successfully managing these local outbreak dynamics.

scholarly journals Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1637
Author(s):  
Solida Long ◽  
Joana B. Loureiro ◽  
Carla Carvalho ◽  
Luís Gales ◽  
Lucília Saraiva ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Tumor Cells ◽  
Mutant P53 ◽  
Wild Type ◽  
Naturally Occurring ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Amino Derivatives ◽  
Carbazole Alkaloids

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.

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