scholarly journals Changes at V2 apex of HIV-1 Clade C trimer enhance elicitation of autologous neutralizing and broad V1V2-scaffold antibodies

2021 ◽  
Author(s):  
Anusmita Sahoo ◽  
Edgar A Hodge ◽  
Celia LaBranche ◽  
Tiffany Turner Styles ◽  
Xiaoying Shen ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Wild Type ◽  
Human Trial ◽  
Specific Antibodies ◽  
Type Protein ◽  
Wild Type Protein ◽  
Clade C ◽  
Intrinsic Dynamic ◽  
Hiv 1

HIV-1 clade C envelope immunogens that elicit both neutralizing and non-neutralizing V1V2-scaffold specific antibodies (protective correlates from RV144 human trial) are urgently needed due to the prevalence of this clade in the most impacted regions worldwide. To achieve this, we introduced structure-guided changes followed by consensus-C sequence-guided optimizations at the V2-region to generate UFO-v2-RQH173 trimer. This improved the abundance of native-like trimers and carried an intrinsic dynamic V2-loop. Following immunization of rabbits, the wild-type protein failed to elicit any autologous neutralizing antibodies but UFO-v2-RQH173 elicited both autologous neutralizing and broad V1V2-scaffold antibodies. The variant with 173Y modification in V2-region, most prevalent among HIV-1 sequences, showed decreased ability in displaying native-like V1V2 epitope with time in-vitro and elicited antibodies with lower neutralizing and higher V1V2-scaffold activities. Our results identify a clade C C.1086-UFO-v2-RQH173 trimer capable of eliciting improved neutralizing and V1V2-scaffold antibodies, and reveal the importance of V2-region in tuning this.

scholarly journals A Novel MVA-Based HIV Vaccine Candidate (MVA-gp145-GPN) Co-Expressing Clade C Membrane-Bound Trimeric gp145 Env and Gag-Induced Virus-Like Particles (VLPs) Triggered Broad and Multifunctional HIV-1-Specific T Cell and Antibody Responses

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 160 ◽  
Author(s):  
Beatriz Perdiguero ◽  
Cristina Sánchez-Corzo ◽  
Carlos Sorzano ◽  
Lidia Saiz ◽  
Pilar Mediavilla ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Hiv Vaccine ◽  
Replication Capacity ◽  
Virus Like Particles ◽  
Modified Vaccinia Virus Ankara ◽  
Clade C ◽  
Membrane Bound ◽  
Hiv 1

The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-β-induced nuclear accumulation

2003 ◽  
Vol 285 (4) ◽  
pp. C823-C830 ◽  
Author(s):  
Bernard A. J. Roelen ◽  
Ori S. Cohen ◽  
Malay K. Raychowdhury ◽  
Deborah N. Chadee ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Phosphorylation Site ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Mutant Protein ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Smad4, the common Smad, is central for transforming growth factor (TGF)-β superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-β and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-β-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

scholarly journals Heparin Induces Harmless Fibril Formation in Amyloidogenic W7FW14F Apomyoglobin and Amyloid Aggregation in Wild-Type Protein In Vitro

PLoS ONE ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. e22076 ◽  
Author(s):  
Silvia Vilasi ◽  
Rosalba Sarcina ◽  
Rosa Maritato ◽  
Antonella De Simone ◽  
Gaetano Irace ◽  
...  
Keyword(s):  
Fibril Formation ◽  
Amyloid Aggregation ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Tusar Kanti Giri ◽  
Tomio Yamazaki ◽  
Núria Sala ◽  
Björn Dahlbäck ◽  
Pablo Garcı́a de Frutos
Keyword(s):  
Recombinant Protein ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Genetic Traits ◽  
Factor Va ◽  
Type Protein ◽  
Wild Type Protein

In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Tusar Kanti Giri ◽  
Tomio Yamazaki ◽  
Núria Sala ◽  
Björn Dahlbäck ◽  
Pablo Garcı́a de Frutos
Keyword(s):  
Recombinant Protein ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Genetic Traits ◽  
Factor Va ◽  
Type Protein ◽  
Wild Type Protein

Abstract In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.

Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility

2002 ◽  
Vol 362 (2) ◽  
pp. 443-451 ◽  
Author(s):  
David BURTON ◽  
Hassan ABDULRAZZAK ◽  
Adam KNOTT ◽  
Kathryn ELLIOTT ◽  
Charles REDWOOD ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Wild Type ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Inhibitory Function ◽  
Type Protein ◽  
Wild Type Protein

We investigated the effects of two mutations in human cardiac troponin I, Arg145 → Gly and Gly203 → Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg145 → Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10–30% [Gly145]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly203 → Ser mutation and troponin C produced less Ca2+-activated force (64±8% of wild-type) and demonstrated lower Ca2+ sensitivity [ΔpCa50 (log of the Ca2+ concentration that gave 50% of maximal activation) 0.25 unit (P < 0.05)] compared with wild-type troponin I, but thin filaments containing [Ser203]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly203 → Ser mutation, or that the disease does not result directly from any particular alteration in contractility.

scholarly journals Identification and Analysis of a Hyperactive Mutant Form of Drosophila P-Element Transposase

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Eileen L Beall ◽  
Matthew B Mahoney ◽  
Donald C Rio
Keyword(s):  
Dna Damage ◽  
Dna Cleavage ◽  
P Element ◽  
In Vitro Assays ◽  
Mutant Form ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI3-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

scholarly journals Tetrahydroxy Cyclic Urea-Potent Inhibitor for HIV-1 Protease Wild Type and Mutant Type—A Computational Design

2008 ◽  
Vol 5 (3) ◽  
pp. 584-592 ◽  
Author(s):  
S. Sree Kanth ◽  
M. Vijjulatha
Keyword(s):  
Biological Activity ◽  
Computational Design ◽  
Protein Docking ◽  
Hydroxyl Group ◽  
Computational Techniques ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Adme Properties ◽  
Hiv 1

A series of novel tetrahydroxy cyclic urea molecules as HIV-1 protease inhibitors were designed using computational techniques. The designed molecules were compared with the known cyclic urea molecules by performing docking studies on six of wild type protein and three mutant protein varieties and calculating their ADME properties. A series of novel molecules were designed by substituting hydrogen at the P1/ P1′ positions with hydroxyl group increasing the bioavailability these had better ADME properties and binding affinity towards HIV-1 protease. The biological activity of these inhibitors were predicted by a model equation generated by the regression analysis between biological activity (log 1/Ki) of known inhibitors and there combined docking scores from six of the wild type protein docking. The synthetic studies are in progress.

Protein S Thr103Asn Mutation Associated with Type II Deficiency Reproduced In Vitro and Functionally Characterised

2000 ◽  
Vol 84 (09) ◽  
pp. 413-419 ◽  
Author(s):  
Tusar Giri ◽  
Pablo de Frutos ◽  
Björn Dahlbäck
Keyword(s):  
Mammalian Cells ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Mutant Protein ◽  
Type Ii ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Functional Defect

SummaryProtein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT-and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa-and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.

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