Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity

2000 ◽  
Vol 381 (7) ◽  
pp. 623-627 ◽  
Author(s):  
Paolo Ascenzi ◽  
Marco Colasanti ◽  
Tiziana Persichini ◽  
Massimo Muolo ◽  
Fabio Polticelli ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Types ◽  
Active Centre ◽  
Human Haemoglobin ◽  
Consensus Motif ◽  
Allosteric Sites ◽  
Great Propensity ◽  
Different Cell Types

Abstract Nitric oxide (NO), produced in different cell types through the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NOreactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acidbase catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2 position has been observed in some NOreactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691 696]. Here, we analyse the threedimensional structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.

scholarly journals CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Douglas K. Fischer ◽  
Akatsuki Saito ◽  
Christopher Kline ◽  
Romy Cohen ◽  
Simon C. Watkins ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Cell Types ◽  
Careful Consideration ◽  
Immunodeficiency Virus ◽  
Cell Cycle Dependence ◽  
Cycle Dependent ◽  
Multiple Strains ◽  
Different Cell Types ◽  
Hiv 1

ABSTRACTThe ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3(>10-fold) and HIV-1LAI(2- to 5-fold) in multiple human cell lines and primary CD4+T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3but not HIV-1LAI. The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAIselected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAIbut not HIV-1NL4-3. The rescue of N57A by G94D in HIV-1LAIis abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3and HIV-1LAICA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types.IMPORTANCEThe specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

New insights into the regulation of inducible nitric oxide synthesis

1994 ◽  
Vol 266 (6) ◽  
pp. E829-E839 ◽  
Author(s):  
S. M. Morris ◽  
T. R. Billiar
Keyword(s):  
Nitric Oxide ◽  
Amino Acid ◽  
Host Response ◽  
Cell Types ◽  
Nitric Oxide Synthesis ◽  
Arginine Metabolism ◽  
Anatomic Site ◽  
Nitrogen Donor ◽  
Potential Impact ◽  
Inducible Nitric Oxide

Recent studies have identified the induction of nitric oxide (NO) synthesis in many cell types as part of the host response to sepsis and inflammation. Induced NO can have a variety of effects which may be detrimental or beneficial during sepsis or inflammation, depending on amount, duration, and anatomic site of synthesis. As arginine is the only physiological nitrogen donor for NO synthesis, metabolism of this amino acid may play an important role in regulation of NO synthesis during sepsis. This review will discuss the roles NO plays in sepsis and the potential impact of arginine metabolism on NO synthesis.

Immunogenicity of novel recombinant human activated factor VII analogues on factor VII neonatally-tolerized rats

2007 ◽  
Vol 98 (10) ◽  
pp. 721-725 ◽  
Author(s):  
Peer Norbert Jørgensen ◽  
Jes Thorn Clausen ◽  
Zaki Salanti ◽  
Lisbeth Bjerring Jensen ◽  
Christian Sommer
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Immune Responses ◽  
Immune Tolerance ◽  
Factor Vii ◽  
Intrinsic Activity ◽  
Active Centre ◽  
Activated Factor Vii ◽  
Significant Difference ◽  
Enzymelinked Immunosorbent Assay

SummaryRecombinant activated factorVII (rFVIIa; NovoSeven®) has been widely used to treat bleeding in patients with haemophilia with inhibitors. To increase the intrinsic activity, analogues of rFVIIa (rFVIIaQ, rFVIIaDVQ, and rFVIIaDVQA) with altered amino acid sequence at or near the active centre have been developed. The immunogenicity of these analogues was tested in a rat immune tolerance model.Neonatal rats received rFVIIa intraperitoneally on post-natal Day 1 and were subsequently challenged with rFVIIa in Freunds Incomplete Adjuvant subcutaneously on Days 10 and 24. Rats were tested for tolerance on Day 32; the tolerant cohort and a parallel cohort of untreated control rats were challenged with rFVIIa, rFVIIaQ, rFVIIaDVQ, or rFVIIaDVQA on Days 46 and 76. Immune responses determined by enzymelinked immunosorbent assay (ELISA) on Day 84 showed no statistically significant difference between the responses in the four control cohorts. Immune responses were higher in the control than in the tolerant cohort. Compared with rFVIIa (4/16), there was no difference in the proportion of rats that broke tolerance following challenge with rFVIIaDVQ (3/16) and rFVIIaDVQA (7/16), whereas a statistically significant greater proportion broke tolerance after challenge with rFVIIaQ (11/16). Therefore, in this model rFVIIaDVQ or rFVIIa DVQA were not more immunogenic than rFVIIa.

scholarly journals Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte

1993 ◽  
Vol 90 (23) ◽  
pp. 11419-11423 ◽  
Author(s):  
I G Charles ◽  
R M Palmer ◽  
M S Hickery ◽  
M T Bayliss ◽  
A P Chubb ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cdna Clone ◽  
Articular Chondrocytes ◽  
Interleukin 1 ◽  
Cell Types ◽  
No Synthase ◽  
Human Chondrocyte ◽  
Oxide Synthase ◽  
Different Cell Types ◽  
Inducible Nitric Oxide

Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.

scholarly journals Differential N-terminal processing of beta and gamma actin in vivo

2021 ◽  
Author(s):  
Li Chen ◽  
Hsin-Yao Tang ◽  
Anna Kashina
Keyword(s):  
Amino Acid ◽  
Physiological Role ◽  
Cell Types ◽  
Amino Acid Sequences ◽  
Major Constituent ◽  
Cytoplasmic Actin ◽  
Mammalian Cell Lines ◽  
Beta Actin ◽  
Different Cell Types

AbstractActin is one of the most essential and abundant intracellular proteins, playing an essential physiological role as the major constituent of the actin cytoskeleton. Two cytoplasmic actins, beta- and gamma-actin, are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-terminus, making it very difficult to dissect their individual regulation in vivo. The majority of actins are N-terminally acetylated, following the removal of N-terminal Met. Here, we analyzed beta and gamma cytoplasmic actin N-termini in vivo and found that beta actin, unlike gamma actin, specifically undergoes sequential removal of N-terminal amino acid Asp residues. This processing affects ∼1-3% of beta actin in different cell types. We identified candidate enzymes capable of mediating this type of processing, and used CRISPR/Cas-9 to delete them, individually or together, in mammalian cell lines. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration, suggesting that the beta actin processing mediated by these enzymes is physiologically important to beta actin function. We propose that selective N-terminal processing of beta actin by sequential removal of Asp contributes to differentiating the functions of non-muscle actin isoforms in vivo.

scholarly journals Structure and tissue-specific expression of the human metallothionein IB gene.

1986 ◽  
Vol 6 (6) ◽  
pp. 2149-2157 ◽  
Author(s):  
A Heguy ◽  
A West ◽  
R I Richards ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Cell Types ◽  
Initiation Site ◽  
Predicted Amino Acid Sequence ◽  
Functional Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
High Level

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-IB is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-IB gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

Structure and tissue-specific expression of the human metallothionein IB gene

1986 ◽  
Vol 6 (6) ◽  
pp. 2149-2157
Author(s):  
A Heguy ◽  
A West ◽  
R I Richards ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Cell Types ◽  
Initiation Site ◽  
Predicted Amino Acid Sequence ◽  
Functional Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
High Level

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-IB is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-IB gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

scholarly journals Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection

2021 ◽  
Vol 11 ◽  
Author(s):  
Diego Rodney Rodrigues de Assis ◽  
Pollyana Maria de Oliveira Pimentel ◽  
Pablo Victor Mendes dos Reis ◽  
Rayane Aparecida Nonato Rabelo ◽  
Ricardo Wagner Almeida Vitor ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Amino Acid Sequence ◽  
Inflammatory Mediators ◽  
Current Treatment ◽  
High Morbidity ◽  
Crude Venom ◽  
Tityus Serrulatus

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides (“Pep1,” “Pep2a,” and “Pep2b”) were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

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