Biomedical subjects

Dimiter S Dimitrov

Publications and source records attributed to Dimiter S Dimitrov.

At least 37 records · Page 2Linked to original sources

PubMed2004

Oligomerization of the SARS-CoV S glycoprotein: dimerization of the N-terminus and trimerization of the ectodomain.

Viral envelope glycoproteins are oligomeric and the quaternary structure is critical for their membrane fusion activity. Typically the transmembrane glycoproteins of class I fusion proteins contain the oligomerization domains and the surface glycoproteins (SU) are monomeric. However, it has been previously demonstrated [J. Biol. Chem. 277 (2002) 19727] that the SU of a murine hepatitis coronavirus (MHV) forms dimers, the dimerization domain overlaps the receptor-binding domain (RBD) and that this dimeric state is important for binding to receptor molecules that initiates entry into cells. We have previously expressed various soluble fragments of the SARS-CoV SU and identified stably folded fragments (residues 272-537) that contain the RBD [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Here, we further characterize these and other fragments in an attempt to identify possible dimerization domains and their role for membrane fusion. We demonstrate that the SU and a shorter 260-amino acid N-terminal fragment (residues 17-276), which folds independently, form dimers. In contrast to the previously characterized MHV SU dimerization, this fragment is upstream and distinct from the RBD. Its deletion abolished S-mediated cell membrane fusion but retained the SU-receptor-binding function indicating the possibility for a role in post-receptor binding steps of the virus entry mechanism. Interestingly, the whole soluble S ectodomain (Se) that contains the dimerization domain but not the transmembrane domain and the cytoplasmic tail forms trimers suggesting the existence of a trimerization domain in the TM subunit in its prefusion state that may lead to a conformation unfavorable for formation of higher-order multimeric structures. These results demonstrate the existence of SU dimers and Se trimers, and indicate the possibility for an unknown mechanism of their role in entry. They also further characterize the S-mediated membrane fusion and could be important for understanding the mechanisms of virus entry, and in the development of therapeutics and vaccines.

Binding Sites↗

PubMed2004

Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120.

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective V(H)-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.

Acquired Immunodeficiency Syndrome↗

PubMed2004

Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope.

The human monoclonal antibody Fab X5 neutralizes a broad range of HIV-1 primary isolates. The crystal structure of X5 has been determined at 1.9 A resolution. There are two crystallographically independent Fab fragments in the asymmetric unit. The crystallographic R value for the final model is 0.22. The antibody-combining site features a long (22 amino acid residues) CDR H3 with a protruding hook-shaped motif. The X5 structure and site-directed mutagenesis data suggest that X5 amino acid residues W100 and Y100F in the CDR H3 motif may be critical for the binding of Fab X5 to gp120. X5 bound to a complex of a CD4 mimetic and gp120 with approximately the same kinetics and affinity as to a CD4-gp120 complex, suggesting that specific interactions between CD4 and X5 are unlikely to contribute to the binding of X5 to gp120-CD4 complexes. Binding of X5 to alanine scanning mutants of gp120JR-CSF complexed with CD4 suggested a critical role of the highly conserved amino acid residues at positions 423 and 432. The X5 structure and fine mapping of its epitope may assist in the elucidation of the mechanisms of viral entry and neutralization, and the development of HIV-1 inhibitors and vaccines.

Antibodies, Monoclonal↗

PubMed2004

A model of the ACE2 structure and function as a SARS-CoV receptor.

The angiotensin-converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system and was very recently identified as a functional receptor for the SARS virus. The ACE2 sequence is similar (sequence identities 43% and 35%, and similarities 61% and 55%, respectively) to those of the testis-specific form of ACE (tACE) and the Drosophila homolog of ACE (AnCE). The high level of sequence similarity allowed us to build a robust homology model of the ACE2 structure with a root-mean-square deviation from the aligned crystal structures of tACE and AnCE less than 0.5A. A prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site. Negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (RBD) of the S-glycoprotein, which we recently identified [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Several distinct patches of hydrophobic residues at the ACE2 surface were noted at close proximity to the charged ridges that could contribute to binding. These results suggest a possible binding region for the SARS-CoV S-glycoprotein on ACE2 and could help in the design of experiments to further elucidate the structure and function of ACE2.

Amino Acid Sequence↗

PubMed2004

A kinetic model of telomere shortening in infants and adults.

We have previously demonstrated that telomeres shorten more rapidly in peripheral mononuclear cells (PBMC) of infants than in adults (Zeichner et al., Blood 93 (1999) 2824). Here we describe a mathematical model that allows quantification of telomere dynamics both in infants and in adults. In this model the dependence of the telomere dynamics on age is accounted by assuming proportionality between the body growth, as approximated by the Gompertz equation, and the increase in the number of PBMCs. The model also assumes the existence of two subpopulations of PBMC with significantly different rates of division. This assumption is based on the results from a previous analysis of in vitro data for telomere dynamics in presence of telomerase inhibitors and our recent data obtained by measurements of BrdU incorporation in T lymphocytes in humans (Kovacs et al., J. Exp. Med. 194 (2001) 1731). The average telomere length of PBMC was calculated as the average length of these two subpopulations. The model fitted our experimental data well and allowed to derive a characteristic time of conversion of the rapidly proliferating cells to slowly proliferating cells on the order of 20 days. The half-life of the slowly proliferating cells was estimated to be about 6 months, which is in good agreement with data obtained by independent methodologies. Comparison of the one-population and two-subpopulations models demonstrated that one population model cannot explain the observed parameters of the terminal restriction fragment (TRF) dynamics while two-subpopulations model does. These results suggest that the rapid telomere shortening in infants is largely determined by the faster PBMC turnover compared to adults. This may have major implications for elucidation of the HIV pathogenesis in infants. One can speculate that the more rapid course of the HIV disease in infants is due to the existence of rapidly dividing cells, which are susceptible to HIV infection. In addition, these results could have implications for understanding of mechanisms of aging.

Adult↗

PubMed2004

Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning.

Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients. However, increasing potency of an antibody by selection from libraries may lead to a decrease in the breadth of neutralization. In an attempt to solve this problem, we subjected a random mutagenesis library of the scFv X5 to sequential rounds of selection on non-homologous HIV-1 envelope glycoproteins (Envs) dubbed sequential antigen panning (SAP). By using SAP, we identified two scFv antibodies, m6 and m9, that were tested with a panel of 33 diverse primary HIV-1 infectious isolates in an assay based on a reporter cell-line expressing high levels of CD4, CCR5 and CXCR4. The IC(50) was less than 50 microg/ml for 21 (m6) and 19 (m9) out of 29 isolates from group M (subtypes A-C, F, G and CRF-01AE) and one isolate from group N; three isolates from group O were not significantly inhibited at 50 microg/ml. The average IC(50) values for the two antibodies were significantly (p<0.001, n=29) lower compared to scFv X5. Their inhibitory activity does not appear to be related to the HIV-1 subtype, coreceptor usage or the disease stage. m9 inhibited infection of peripheral blood mononuclear cells by the primary isolates JRCSF, 89.6 and BR020 with IC(90) of 4, 6 and 25 microg/ml, respectively; for a single-round infection by pseudovirus, the IC(90) for JRSCF, 89.6, YU2 and HXBc2 was 15, 5, 15 and 5 microg/ml, respectively. In these two assays the IC(90) for m9 was, on average, two- to threefold lower than for scFv X5. These results demonstrate that both the potency and the breadth of HIV-1 neutralization of one of the few known potent broadly cross-reactive human monoclonal antibodies, scFv X5, could be improved significantly. However, only experiments in animal models and clinical trials in humans will show whether these new scFvs and the approach for their identification have potential in the development of prophylactics and therapeutics for HIV-1 infections.

Antibody Affinity↗

PubMed2004

Identification of a novel CD4i human monoclonal antibody Fab that neutralizes HIV-1 primary isolates from different clades.

A new human monoclonal antibody (hmAb), designated m16, was selected by sequential antigen panning (SAP) of a human phage display library against recombinant soluble HIV-1 envelope glycoproteins (Envs) (gp140s) and their complexes with soluble CD4. It bound with high (nM) affinity to gp120 and gp140; the binding was further enhanced by interactions of the Envs with CD4. m16 inhibited cell fusion mediated by the Envs of 9 HIV-1 isolates from clades A, B, E and G with potency on average comparable to that of the broadly neutralizing human monoclonal antibody Fab X5. The identification of a new hmAb with broad neutralizing activity that exhibits differential inhibitory profile suggests a potential for its use as a component of anti-HIV-1 treatments.

Antibodies, Monoclonal↗

PubMed2004

Virus entry: molecular mechanisms and biomedical applications.

Viruses have evolved to enter cells from all three domains of life--Bacteria, Archaea and Eukaryotes. Of more than 3,600 known viruses, hundreds can infect human cells and most of those are associated with disease. To gain access to the cell interior, animal viruses attach to host-cell receptors. Advances in our understanding of how viral entry proteins interact with their host-cell receptors and undergo conformational changes that lead to entry offer unprecedented opportunities for the development of novel therapeutics and vaccines.

Animals↗

PubMed2004

Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody.

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.

Alanine↗

PubMed2003

The SARS-CoV S glycoprotein: expression and functional characterization.

We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S(e), were not cleaved to any significant degree. They ran at about 180-200kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17-537 and 272-537 but not 17-276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450-454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors.

Animals↗

PubMed2003

The secret life of ACE2 as a receptor for the SARS virus.

The membrane-associated carboxypeptidase angiotensin-converting enzyme 2 (ACE2) is an essential regulator of heart function. Now, Li at al. identify and characterize an unexpected second function of ACE2 as a partner of the SARS-CoV spike glycoprotein in mediating virus entry and cell fusion.

Angiotensin-Converting Enzyme 2↗

PubMed2003

Purified complexes of HIV-1 envelope glycoproteins with CD4 and CCR5(CXCR4): production, characterization and immunogenicity.

The ability to readily elicit broadly neutralizing antibodies to HIV-1 remains elusive. We and others have hypothesized that interaction of the viral envelope glycoprotein (Env, gp120-gp41) with its receptor molecules could enhance the exposure of conserved epitopes that may facilitate the elicitation of broadly neutralizing antibodies. The Env-CD4-coreceptor complexes mediate HIV-1 entry into cells and serve as a major target for inhibitors of this process. To begin to evaluate their potential also as vaccine immunogens we produced relatively large amounts of complexes of purified recombinant soluble truncated Env, gp140(89.6) or gp120(89.6), with CD4 and CCR5 or CXCR4. We found that gp140(gp120)-CD4-CCR5 complexes are stable and immunogenic in mice transgenic for human CD4 and CCR5. They elicited anti-gp120 and anti-gp140 antibodies that inhibited an heterologous primary HIV-1 isolate (JR-FL) with two- to threefold higher neutralizing activity than those elicited by gp120 and gp140. The antibodies elicited by the complexes competed better with the antibodies X5 and CG10 but not with b12 for binding to gp120 and gp120-CD4 complexes compared to those elicited with gp140(120) alone. These findings suggest that stable purified Env-CD4-CCR5(CXCR4) complexes can be produced in relatively large amount sufficient for their further characterization that may help in the development of novel vaccines candidates.

Animals↗

PubMed2003

Evaluation of gene expression measurements from commercial microarray platforms.

Multiple commercial microarrays for measuring genome-wide gene expression levels are currently available, including oligonucleotide and cDNA, single- and two-channel formats. This study reports on the results of gene expression measurements generated from identical RNA preparations that were obtained using three commercially available microarray platforms. RNA was collected from PANC-1 cells grown in serum-rich medium and at 24 h following the removal of serum. Three biological replicates were prepared for each condition, and three experimental replicates were produced for the first biological replicate. RNA was labeled and hybridized to microarrays from three major suppliers according to manufacturers' protocols, and gene expression measurements were obtained using each platform's standard software. For each platform, gene targets from a subset of 2009 common genes were compared. Correlations in gene expression levels and comparisons for significant gene expression changes in this subset were calculated, and showed considerable divergence across the different platforms, suggesting the need for establishing industrial manufacturing standards, and further independent and thorough validation of the technology.

Cell Line↗

PubMed2003

Cell biology of virus entry: a review of selected topics from the 3rd International Frederick meeting.

Following the first two Frederick meetings on virus entry in 1997 [Cell 91 (1997) 721](1) and in 2000 [Cell 101 (2000) 697](2), further developments in our understanding of the multifactorial and multistage process of virus entry, and possible biomedical implications were presented and discussed in a lively fashion by leading scientists from around the world at the third Frederick meeting on the Cell Biology of Viral Entry (May 7-10, Frederick, MD) organized by R. Blumenthal (NCI-Frederick, NIH, Frederick) and E. Hunter (University of Alabama, Birmingham). Unlike the previous two meetings, non-enveloped viruses were not discussed this time, and the focus was how envelope glycoproteins (Envs) mediate entry into cells. Major topics included Env structure, virus receptors, entry intermediates, membrane fusion, fusion kinetics, and rafts. Virus envelope structures will be described in more detail here because the other topics are extensively discussed in the other chapters of this volume.

Biological Transport↗

PubMed2003

Evidence for CD4-enchanced signaling through the chemokine receptor CCR5.

The chemokine receptor CCR5 is constitutively associated with the T cell co-receptor CD4 in plasma cell membranes, but the physiological role of this interaction has not been elucidated. Here we show that detergent-solubilized, purified CCR5 can directly associate with purified soluble fragments of the extracellular portion of CD4. We further demonstrate that the physical association of CCR5 and CD4 in membrane vesicles results in the formation of a receptor complex that exhibits macrophage inflammatory protein 1beta (MIP-1beta) binding properties that are distinct from CCR5. The affinity of the CD4-CCR5 complex for MIP-1beta was 3.5-fold lower than for CCR5, but the interaction of CD4 and CCR5 resulted in a receptor complex that exhibited enhanced G-protein signaling as compared with CCR5 alone. MIP-1beta-induced G-protein activation was further increased by simultaneous stimulation of CD4 with its natural agonist, interleukin-16. Thus, the physical association of CD4 and CCR5 results in receptor cross-talk with allosteric CD4-dependent regulation of the binding and signaling properties of CCR5. Although the precise physiological role of the CD4 effects on CCR5-mediated signaling remains unknown, one can speculate that the cross-talk is a component of mechanisms involved in the fine tuning of immune system cell responses.

CD4 Antigens↗

PubMed2003

Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library.

Identification of broadly cross-reactive human monoclonal antibodies (mAbs) has major implications for development of vaccines, inhibitors and research tools. Here we describe a sequential antigen panning (SAP) methodology that may facilitate the selection of such antibodies. An HIV-specific antibody Fab (m18) was selected from a human Fab phage-display library by SAP against several recombinant soluble HIV envelope glycoproteins (Envs) and Env-sCD4 complexes. This Fab bound to a variety of recombinant soluble Envs (gp140s) from primary HIV isolates representing different clades, and inhibited cell fusion and virus entry mediated by Envs of primary HIV isolates. The methodology and the results may have implications for development of HIV vaccines and inhibitors, as well as for identification of antibodies to conserved epitopes on rapidly mutating viruses and cells.

Antibodies, Monoclonal↗

PubMed2003

Role of Ets/Id proteins for telomerase regulation in human cancer cells.

Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms. Recently, we have proposed that Ets proteins may be important for regulation of telomerase activity in leukemic cells. Here we provide further evidence for the role of Ets family members and related Id proteins in telomerase regulation and characterize the underlying molecular mechanisms. By using PCR-based and gel shift assays we demonstrated specific binding to a core hTERT promoter of Ets2, Fli1, Id2, c-Myc, Mad1, and Sp1 in lysates from subclones of U937 cells. Further analysis of binding of purified proteins and various mutants of the hTERT promoter suggested the existence of a trimolecular Ets-Id2-DNA complex, and Ets inhibitory activity mediated by c-Myc and the Ets binding site on the core hTERT promoter at -293 bp from the transcription initiation site as well as a positive Ets regulatory effect mediate through another Ets binding site at -36 bp. This analysis provided evidence for the existence of negative and positive Ets regulatory site and suggested a complex interplay between Ets/Id family members and c-Myc that may be an important determinant of the diversity of telomerase activity in leukemia and other cancers.

DNA-Binding Proteins↗

PubMed2003

Cancer treatment by telomerase inhibitors: predictions by a kinetic model.

The inhibition of telomerase activity in actively dividing cells leads to shortening of their telomeres and suppression of cell growth when the telomere lengths become smaller than a certain threshold value (typically about 1-2 kb of DNA). We evaluated the time (efficacy delay) required to reach the threshold telomeric DNA size after initiation of treatment, which is of critical importance for the efficacy of telomerase inhibitors. A model based on the solution of a system of differential equations was developed to analyze the efficacy delay and dynamics of tumor growth. The efficacy delay was strongly dependent on the size distribution of telomere lengths at the treatment initiation. An increase in the heterogeneity of telomere size resulted in shortening of the delay. However, the long-term dynamics of tumors with homogeneous populations of telomeres were more significantly affected by telomerase inhibitors compared to tumors with heterogeneous size distribution of telomeres. Size distribution of telomeres and tumor doubling times are of critical importance for the dynamics of tumor growth in presence of telomerase inhibitors.

Cell Division↗