scholarly journals Transglutaminase 5 is regulated by guanine–adenine nucleotides1

2004 ◽  
Vol 381 (1) ◽  
pp. 313-319 ◽  
Author(s):  
Eleonora CANDI ◽  
Andrea PARADISI ◽  
Alessandro TERRINONI ◽  
Valentina PIETRONI ◽  
Sergio ODDI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Cross Linking ◽  
Bifunctional Enzyme ◽  
Amino Acid Sequence Alignment ◽  
Gtp Binding ◽  
Epidermal Tissue ◽  
Isopeptide Bonds

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

scholarly journals In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

2018 ◽  
Vol 115 (24) ◽  
pp. E5477-E5486 ◽  
Author(s):  
Chungyu Chang ◽  
Brendan R. Amer ◽  
Jerzy Osipiuk ◽  
Scott A. McConnell ◽  
I-Hsiu Huang ◽  
...  
Keyword(s):  
Cross Linking ◽  
Structural Elements ◽  
Gram Positive Bacteria ◽  
Activating Mutations ◽  
In Vitro Reconstitution ◽  
Isopeptide Bonds ◽  
Signature Sequence ◽  
Class C ◽  
Enzyme Intermediate

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2Msynthesizes pilus fibers with correct Lys–Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA–SrtA–SpaA polymerization intermediate depicts SrtA2Msandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2Mcan terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

scholarly journals Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene

2010 ◽  
Vol 95 (3) ◽  
pp. 1301-1308 ◽  
Author(s):  
Susanne Bens ◽  
Angelika Mohn ◽  
Bilgin Yüksel ◽  
Alexandra E. Kulle ◽  
Matthias Michalek ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Adrenal Hyperplasia ◽  
Turkish Patient ◽  
Star Protein ◽  
Sex Development ◽  
Congenital Lipoid Adrenal Hyperplasia

Abstract Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia. Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient). Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the α4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein. Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient’s phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

scholarly journals Novel MAPK/AKT-impairing germline NRAS variant identified in a melanoma-prone family

2021 ◽  
Author(s):  
Kevin M. Brown ◽  
Mai Xu ◽  
Michael Sargen ◽  
Hyunbum Jang ◽  
Mingzhen Zhang ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Binding Pocket ◽  
Pi3k Pathway ◽  
Melanoma Risk ◽  
Mediterranean Populations ◽  
Risk Genes ◽  
1000 Genomes ◽  
Gtp Binding ◽  
The Us

AbstractWhile several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations, we identified a novel NRAS variant (c.170A>C, p.D57A) in a melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11 273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2+ binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A>C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.

scholarly journals Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

2014 ◽  
Vol 89 (1) ◽  
pp. 208-219 ◽  
Author(s):  
Jiong Shi ◽  
Jing Zhou ◽  
Upul D. Halambage ◽  
Vaibhav B. Shah ◽  
Mallori J. Burse ◽  
...  
Keyword(s):  
Amino Acid ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Binding Pocket ◽  
Host Protein ◽  
Viral Capsid ◽  
Inhibitory Protein ◽  
Hiv 1 ◽  
Ca Protein

ABSTRACTThe HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexesin vitroand permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.IMPORTANCEThe HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

scholarly journals Effect of Various Electrolytes on Theophylline Loaded Sodium Alginate Beads Prepared by Ionic Cross Linking Technique

2013 ◽  
Vol 11 (2) ◽  
pp. 181-189
Author(s):  
Sheikh Tasnim Jahan ◽  
Sams Mohammad Anowar Sadat ◽  
Muhammad Rashedul Islam ◽  
ATM Zafrul Azam ◽  
Jakir Ahmed Chowdhury
Keyword(s):  
Drug Release ◽  
Sodium Alginate ◽  
Electrolyte Concentration ◽  
Research Work ◽  
Three Dimensional ◽  
Alginate Beads ◽  
Water Soluble ◽  
Cross Linking ◽  
Bonding Structure

The purpose of t h e present research work was to prepare alginate beads containing water soluble drug theophylline using ionic cross linking technique, with electrolyte type and concentration as variables. In this study, the beads were characterized and evaluated in respect of their surface morphology, swelling index and in vitro kinetics. The comparative study among the three polyvalent cationic cross linking agents CaCl2 , BaCl2 and Al2 (SO4)3 were investigated based on their cationic charges. Divalent cation, Ca2+ and Ba2+ containing beads showed simultaneous decrease in drug release with increasing electrolyte amount. In case of Al3+ -alginate beads, the delay in release was due to the ability of Al3+ to form three dimensional bonding structure with the sodium alginate inside the beads. As a result, swelling of beads is delayed leading to slow disintegration. Scanning electron microscope (SEM) photomicrographs revealed that with the increase in the electrolyte concentration the density of the cross link is also increased. When the electrolyte concentration is 5 % then the beads surface is rough and rod shape drug is visible. But when the electrolyte concentration is increased from 10 % to 15 % the surface is comparatively smoother and both the swelling property and in vitro drug release are decreased. Most of the formulations followed Higuchi drug release model. DOI: http://dx.doi.org/10.3329/dujps.v11i2.14578 Dhaka Univ. J. Pharm. Sci. 11(2): 181-189, 2012 (December)

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

2014 ◽  
Vol 70 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Sonia Fieulaine ◽  
Michel Desmadril ◽  
Thierry Meinnel ◽  
Carmela Giglione
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Human Counterpart ◽  
Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

scholarly journals Additive and epistatic effects influence spectral tuning in molluscan retinochrome opsin

2021 ◽  
Author(s):  
G Dalton Smedley ◽  
Kyle E McElroy ◽  
Jeanne M Serb
Keyword(s):  
Amino Acid ◽  
Blue Shift ◽  
Binding Pocket ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Spectral Tuning ◽  
Closely Related Species ◽  
Absorbance Peak ◽  
The Relationship

The relationship between genotype and phenotype is nontrivial due to often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype (λmax). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket are responsible for fine spectral tuning, or small changes in the λmax of these light sensitive proteins. Most mutations caused a blue shift regardless of the retinochrome background, with shifts ranging from a 12 nm blue shift to a 5 nm red shift from the wild-type λmax. These mutations do not show an additive effect, but rather suggests the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis.

scholarly journals Generation of guanine–amino acid cross-links by a free radical combination mechanism

2014 ◽  
Vol 16 (23) ◽  
pp. 11729-11736 ◽  
Author(s):  
Yuriy Uvaydov ◽  
Nicholas E. Geacintov ◽  
Vladimir Shafirovich
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Side Chain ◽  
Cross Linking ◽  
Two Photon ◽  
Ionization Method ◽  
Cross Links ◽  
Photon Ionization ◽  
Amino Acid Radicals

The key step of DNA–protein cross-linking in vitro is the combination of guanine neutral radicals with side-chain C-centered amino acid radicals produced by a two-photon ionization method.

scholarly journals MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters

2003 ◽  
Vol 23 (5) ◽  
pp. 1656-1665 ◽  
Author(s):  
Francesca Lembo ◽  
Raffaela Pero ◽  
Tiziana Angrisano ◽  
Carmen Vitiello ◽  
Rodolfo Iuliano ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Interacting Protein ◽  
Amino Acid Region ◽  
Gtp Binding ◽  
Acid Region

ABSTRACT We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.

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