Human immunodeficiency virus (HIV) gp41 escape mutants: cross-resistance to peptide inhibitors of HIV fusion and altered receptor activation of gp120

Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.     

Fate of predator and prey proteins during growth of Bdellovibrio bacteriovorus on Escherichia coli and Pseudomonas syringae prey

A two-dimensional electrophoretic analysis of protein distribution followed by identification of selected proteins by mass spectrometry was performed on fresh bdellovibrio cultures containing attack phase cells of the predatory bacterium Bdellovibrio bacteriovorus strain 109J-1 and the remains of an Escherichia coli or a Pseudomonas syringae pv. tomato prey. Cleavage of the peptidoglycan-associated outer membrane proteins (OMPs) OmpA in E. coli and OprF in P. syringae occurred in both prey. The tryptic peptides obtained from the cleavage products of OmpA and OprF were all located within the 19-kDa pronase-resistant N-terminal parts of the corresponding proteins. The predator cell fraction was separated from the prey ghosts in fresh bdellovibrio cultures by centrifugation on a Percoll-sucrose cushion. Proteins from each fraction were separated by two-dimensional electrophoresis and identified by mass spectrometric analysis. As no prey OMP could be detected in the predator cell fraction, it was concluded that prey OMPs are not transferred to the predator, as had been suggested previously. However, a protein from the predator was found bound to ghost cell envelopes. This protein may correspond to a protein earlier suggested to be associated with the prey outer or cytoplasmic membranes. Along with recently described polypeptides from B. bacteriovorus strains 100 and 114, it forms a new family of putative outer membrane proteins.     

Dendrite-like process formation and cytoskeletal remodeling regulated by delta-catenin expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of delta-catenin (or NPRAP/Neurojungin), a member of p120(ctn) subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of delta-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. delta-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of delta-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When delta-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that delta-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.     

Changes in the Level of DNA Damage in Mouse Cells Induced by Atmospheric Factors

Biophysics - Abstract—Changes in the level of DNA damage induced by atmospheric factors were evaluated from the percentage of tail DNA (%TDNA) in the comet assay of mouse cells for blood...     

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4′) match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.Several molecular mechanisms are in place to combat transformed malignant cells and virally infected cells. Granzymes (Gr)¹, a family of structurally related serine proteases found in the granules of many immune cells, play crucial roles in such cellular defense mechanisms. The granzyme family consists of five human proteases (granzymes A, B, H, K, and M) and 10 murine members (granzymes A to G, K, M, and N). To date for four human granzymes (A, B, K, and M) clear murine orthologs have been assigned, while the most probable murine ortholog of human granzyme H (GrH) is granzyme C (GrC) based on their 70% sequence similarity, 61% sequence identity, and identical chromosomal location relative to granzyme B (GrB). Furthermore, both granzymes are expressed by NK and CD4+ T-cells (1, 2): GrH is constitutively expressed at high levels in NK cells and less in CD4+ T cells, whereas GrC expression can be observed after stimulation. Thus, their overlapping expression profiles further support possible functional similarities between mouse GrC and human GrH.The physiological role of granzymes was presumed to be the induction of death in target cells. Granzyme B is a highly efficient cytotoxin (3) and other granzymes such as GrA, GrC, GrF, and GrK can cause cell death at high concentrations (4–8). Recently, granzymes A, K, and M were shown to steer inflammatory processes when used at physiological levels (9–11). Two previous studies identified GrH as an alternative cytotoxic effector protease. Although both studies showed typical hallmarks of apoptosis, including mitochondrial depolarization, reactive oxygen species (ROS) production, DNA degradation as well as chromatin condensation, Fellows et al. (12) found that, in contrast to GrB mediated cell death, GrH induced cell death did not result in caspase activation, cytochrome c release, or cleavage of Bid and/or ICAD. In sharp contrast however, Hou et al. (13) demonstrated that GrH induced apoptosis depended on caspase activation and that GrH cleaved ICAD and Bid, the latter ultimately resulting in mitochondrial cytochrome c release. Such discrepancies have been documented in other granzyme studies and are usually linked to the use of different granzyme delivery systems, sources of recombinantly produced granzymes, differences in the granzyme concentration and species-specific differences in substrate specificities (14).GrC induces cell death reminiscent of GrH induced cell death as observed by Fellows et al. (12), as both exert their cytotoxic functions independent of caspase activation, Bid or ICAD cleavage, or by mitochondrial release of cytochrome c (5). GrC induced apoptosis was characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. Supporting evidence implying a role for GrC in lymphocyte induced cytotoxicity was inferred from the fact that GrB cluster-deficient mice (mice that do not express GrB and show a five- to sixfold reduced expression of GrC and GrF respectively) display a more pronounced defect in the clearance of allogeneic tumor cells when compared with GrB-only knockout mice (4). This suggests that GrC and/or GrF may be important for correct functioning of cytotoxic lymphocytes. Besides, in GrB-only knockout mice, a likely compensatory mechanism occurs, given that during cytotoxic lymphocyte activation, peak expression of GrC occurs earlier, giving rise to overall higher GrC levels as compared with wild-type mice (1, 4, 15). Of note, despite its implication in cytotoxicity, GrC was shown to be an inefficient cytotoxin, with a 2900-fold greater EC₅₀ value as compared with hGrB when delivered into P815 cells with recombinant mouse perforin (16).Positional Scanning Synthetic Combinatorial Libraries (PS-SCL) revealed the chymotrypsin-like activity of GrH, which it shares with granzyme M (17–19), with an optimal P4-P1 peptide substrate sequence Pro-Thr-Ser-Tyr. Less stringent specificities were observed at positions P4, P3, and P2 (19) where GrH seems to tolerate multiple amino acids with different chemical characteristics (especially neutral and aliphatic amino acids). Although GrH and GrM (optimal peptide identified as Lys-Val-Pro-Leu) share a P1 chymotryptic activity, GrH prefers bulkier, aromatic amino acids (Tyr and Phe) at P1 whereas GrM prefers Leu. Both further recognize Leu and Met at P1, implying that some substrates could be cleaved by both granzymes. GrM also shows broader specificities at P3, but at P4 and P2 it prefers basic residues and Pro respectively. Besides, GrC chymase activity could be inferred from N-terminal COFRADIC and substrate phage display screens, which defined the P4-P3′ substrate specificity of GrC as [Ile/Val]-X-[Phe-Tyr]-[Phe-Leu-Tyr-Met]↓X-[Gly-Ser]-[Asp-Glu] (16).Recently, the crystal structure of the D₁₀₂>N GrH variant in complex with a decapeptide substrate (PTSYAGDDSG) or inhibitor (Ac-PTSY-chloromethylketone) was resolved (20). The electron density maps clearly showed the full length protease adopting a canonical structure of 2 α-helices and 13 β-strands assembled into two juxtaposed β-barrel domains bridged by the catalytic triad (composed of His₅₇, Asp₁₀₂ and Ser₁₉₅). The S1 specificity pocket is built up from residues of 2 loops; loop 189 (from residue 183–196) and loop 220 (from residue 215–226) with the determinants being Thr₁₈₉, Gly₂₁₆, and Gly₂₂₆. From these, Gly₂₂₆ was assigned as the most important determinant for the preference of bulky aromatic amino acids (such as Tyr and Phe) at P1. Where GrH contains a Gly at position 226, GrB, GrC, and GrM harbor an Arg, Gln, and Pro residue respectively (supplemental Fig. S1). Mutation of Gly₂₂₆ in GrH to Arg₂₂₆ compromised binding of bulky aromatic residues and enabled interaction with negatively charged amino acids. Hydrogen bond formation between a P1 Tyr and Asn₂₁₇ of GrH could furthermore strengthen the observed preference of Tyr over Phe at P1 (19). The presence of Pro at position 226 in GrM instead of Gly narrows the S1 pocket and might be indicative for the preference of Leu instead of Phe and Tyr at P1. In addition, the structure revealed that the S4′ pocket formed by the backbones of the Arg₃₉-Lys₄₀-Arg₄₁ motif resulted in a preference for acidic residues at P4′, in addition to influencing the P3′ preference for acidic residues via salt bridge formation with this Lys₄₀. Although the Arg₃₉-Lys₄₀-Arg₄₁ motif is a unique GrH feature, GrB possesses a partially degenerate Leu-Lys-Arg motif in which Lys₄₀ also enables interaction with acidic residues in P3′. Next to P3′, proteome-wide screening for GrB substrates led to the identification of a clear preference for acidic residues at P4′ caused by salt bridge formation with Arg₄₁ (21), a characteristic that was previously assigned to Lys₄₀ of GrB (22). To validate these structural observations, similar to the P4′ Asp mutation in the GrB substrate PI-9 (22), both Asp residues found at P3′ and P4′ in the nuclear phosphoprotein La, previously identified as a macromolecular GrH substrate (23), were mutated to Ala. These mutations completely abolished GrH mediated proteolysis, a first indication that the P4-P1 specificity profile is not a sole determinant for substrate recognition by GrH. Next to the La phosphoprotein, cleavage of the viral adenovirus DNA binding protein (DBP) and the 100K assembly protein (L4–100K), the latter resulting in the relieve of GrB inhibition by L4–100K, has been observed, implicating GrH in host antiviral defense and indicative for a functional synergism between GrH and GrB (24).Elucidation of the crystal structure, with an electron density map showing residues Ile₁₆-His₂₄₄ (i.e. 94% of full length GrC and 99.6% of active GrC), furthermore showed that wild-type GrC can be restrained in its proteolytic function despite the presence of the catalytic triad residues His₅₇-Asp₁₀₂-Ser₁₉₅ (16). Comparing the crystal structures of GrC and GrA showed an unusual conformation of the active site, which could explain the inactivity of GrC. Apparently, the 190-strand, preceding Ser₁₉₅ of the catalytic triad, has undergone a register shift on the structural level leading to Phe₁₉₁ filling the S1 pocket. This pocket is furthermore covered by Glu₁₉₂, which forms a salt bridge with Arg₉₉ and a hydrogen bond with the backbone amide of Ser₁₉₅. Because of this unusual conformation, the Glu₁₉₂-Glu₁₉₃ peptide bond points away from the substrate, as such leading to an improperly formed oxyanion hole, which normally stabilizes the negatively charged substrate oxygen atom during catalysis. Mutation of Glu₁₉₂-Glu₁₉₃ to the corresponding amino acids in its closest related homolog GrB (Arg₁₉₂-Gly₁₉₃) disrupted the Glu₁₉₂-Ser₁₉₅ hydrogen bond and caused a shift of the 190-strand, thereby clearing the S1 pocket and giving rise to an active GrC mutant. These results indicate allosteric control of wild-type GrC in which binding of a substrate or cofactor might stabilize the 190-strand, which becomes extremely mobile due to breaking the Glu₁₉₂-Ser₁₉₅ hydrogen bond and turning the region surrounding the active site more rigid.To elucidate a possible functional homology between GrH and GrC, we performed differential degradome analyses using N-terminal COFRADIC in the species-matching proteome backgrounds. For these analyses, we made use of the active E₁₉₂E₁₉₃>RG GrC mutant as described in (16) (further referred to as mut GrC). These analyses, further complemented with phage display data on granzyme H, clearly show that both granzymes display a highly similar substrate specificity profile, analogous to other orthologous granzymes. Besides, and in contrast to GrA, a general conservation of the extended substrate specificity profiles among the homologous granzymes B, C, H, and M could be observed across species, highlighting the importance of the extended substrate specificity in steering substrate cleavage susceptibility.     

Purification of the channel component of the mitochondrial calcium uniporter and its reconstitution into planar lipid bilayers

The purification of the channel-forming component of the mitochondrial calcium uniporter and its channel properties are described. After ethanol and 50% ethanol-water extraction of mitochondria from beef heart or perfused rat liver, the extract was passed through thiopropyl-Sepharose 6B column, and absorbed components were eluted with 2-mercaptoethanol, followed by gel-filtration on Sephadex G-15. The last fraction eluted (M _r about 2000) was then subjected to reverse-phase high-performance liquid chromatography. Of the more than 10 distinct peaks, only one showed specific Ca²⁺-channel activity in BLM with properties similar to earlier, less extensively purified preparations, i.e., conductance of 20 pS and multiples thereof, clustering of channels, participation of 2 or more subunits in channel formation, and sensitivity to 1 µM ruthenium red. Voltage sensitivity and cooperativity between channels are described. The Ca²⁺-binding glycoprotein with which the peptide was associated was found to have high homology with human acid ₁-glycoprotein (orosomucoid) and to show identity with beef plasma orosomucoid in the Ouchterlony immunodiffusion test.     

A preliminary simulation model of movement of larvae ofCulex pipiens molestus (Diptera: Culicidae) (Experimental studies on the dispersal of insects,II)

Experiments for analyzing the movement and dispersal of larvae of C. pipiens molestus were made and preliminaly simulation models were constructed. The models were based on two distributions, the distance and direction of movements between 1 minute intervals. The distribution of distance is represented by a unimodal curve, and that of angle by a bimodal one. The results of simulation reasonably depicted the actual process of movements. Nevertheless, the simulated time-dispersal curves overestimated the number of dispersed individuals. This might be due to mutual attraction among the larvae.     

Non-synonymous and synonymous coding SNPs show similar likelihood and effect size of human disease association

Many DNA variants have been identified on more than 300 diseases and traits using Genome-Wide Association Studies (GWASs). Some have been validated using deep sequencing, but many fewer have been validated functionally, primarily focused on non-synonymous coding SNPs (nsSNPs). It is an open question whether synonymous coding SNPs (sSNPs) and other non-coding SNPs can lead to as high odds ratios as nsSNPs. We conducted a broad survey across 21,429 disease-SNP associations curated from 2,113 publications studying human genetic association, and found that nsSNPs and sSNPs shared similar likelihood and effect size for disease association. The enrichment of disease-associated SNPs around the 80(th) base in the first introns might provide an effective way to prioritize intronic SNPs for functional studies. We further found that the likelihood of disease association was positively associated with the effect size across different types of SNPs, and SNPs in the 3' untranslated regions, such as the microRNA binding sites, might be under-investigated. Our results suggest that sSNPs are just as likely to be involved in disease mechanisms, so we recommend that sSNPs discovered from GWAS should also be examined with functional studies.     

Identifying a high fraction of the human genome to be under selective constraint using GERP++

Computational efforts to identify functional elements within genomes leverage comparative sequence information by looking for regions that exhibit evidence of selective constraint. One way of detecting constrained elements is to follow a bottom-up approach by computing constraint scores for individual positions of a multiple alignment and then defining constrained elements as segments of contiguous, highly scoring nucleotide positions. Here we present GERP++, a new tool that uses maximum likelihood evolutionary rate estimation for position-specific scoring and, in contrast to previous bottom-up methods, a novel dynamic programming approach to subsequently define constrained elements. GERP++ evaluates a richer set of candidate element breakpoints and ranks them based on statistical significance, eliminating the need for biased heuristic extension techniques. Using GERP++ we identify over 1.3 million constrained elements spanning over 7% of the human genome. We predict a higher fraction than earlier estimates largely due to the annotation of longer constrained elements, which improves one to one correspondence between predicted elements with known functional sequences. GERP++ is an efficient and effective tool to provide both nucleotide- and element-level constraint scores within deep multiple sequence alignments.     

Ability of ELISA and a toxin neutralization assay to detect changes in immunogenicity of a recombinant Bacillus anthracis protective antigen vaccine upon storage

We examined the capability of a mouse immunogenicity assay to detect improper storage of a recombinant protective antigen (rPA)-based anthrax vaccine formulated with an aluminum adjuvant, using ELISA and a toxin neutralization assay (TNA) to measure the antibody response to rPA. The vaccine was stored at 4 °C, room temperature (RT) or 37 °C for one, four and eight weeks and used for immunization, along with freshly prepared vaccine. Results showed that, contrary to ELISA, TNA is suitable to detect a loss of immunogenicity of the rPA vaccine following its exposure to RT for a period of eight weeks and to 37 °C for a period as short as 1 week.