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John Jeff Alvarado

Bio: John Jeff Alvarado is an academic researcher from University of Pittsburgh. The author has contributed to research in topics: Proto-oncogene tyrosine-protein kinase Src & SH3 domain. The author has an hindex of 7, co-authored 11 publications receiving 192 citations. Previous affiliations of John Jeff Alvarado include Albert Einstein College of Medicine.

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Journal ArticleDOI
TL;DR: An assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein is developed and a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants.

49 citations

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TL;DR: The results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners.

38 citations

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TL;DR: Comparison of microsporidian Met AP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporaidiosis, particularly in patients that are immunosuppressed.

30 citations

Journal ArticleDOI
TL;DR: The crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hckprotein.

30 citations

Journal ArticleDOI
TL;DR: Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
Abstract: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.

23 citations


Cited by
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Journal ArticleDOI
TL;DR: An overview of the poly-l-proline type II (PPII) helix and of the place it holds in the current understanding of protein structure and function is given.

419 citations

Journal ArticleDOI
TL;DR: This review mainly focuses on the existence of isoxazoline derivatives in natural sources, their isolation and uses there of as anticancer agents besides highlighting the synthetic pathways to achieve these compounds.

190 citations

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TL;DR: It was shown that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumgeillin.
Abstract: Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2) in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.

155 citations

Journal ArticleDOI
TL;DR: The possibility of inhibiting the HIV-1 LTR promoter by G-quadruplex-interacting small molecules is disclosed, providing a new pathway to development of anti-HIV-1 drugs with unprecedented mechanism of action.
Abstract: G-Quadruplexes, noncanonical nucleic acid structures, act as silencers in the promoter regions of human genes; putative G-quadruplex forming sequences are also present in promoters of other mammals, yeasts, and prokaryotes Here we show that also the HIV-1 LTR promoter exploits G-quadruplex-mediated transcriptional regulation with striking similarities to eukaryotic promoters and that treatment with a G-quadruplex ligand inhibits HIV-1 infectivity Computational analysis on 953 HIV-1 strains substantiated a highly conserved G-rich sequence corresponding to Sp1 and NF-κB binding sites Biophysical/biochemical analysis proved that two mutually exclusive parallel-like intramolecular G-quadruplexes, stabilized by small molecule ligands, primarily fold in this region Mutations disrupting G-quadruplex formation enhanced HIV promoter activity in cells, whereas treatment with a G-quadruplex ligand impaired promoter activity and displayed antiviral effects These findings disclose the possibility of inhibiting th

144 citations

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TL;DR: This Review catalogs applications of HDX MS that have appeared in the literature during the 30 months from January 2012 to June 2014 as penetration of the method into nonacademic sectors where confidentiality is necessary is also at an all-time high.
Abstract: Hydrogen/deuterium exchange (HDX) detected by mass spectrometry (MS) is extraordinarily useful in the study of many aspects of proteins, especially the analysis of protein conformation and dynamics. While once a challenging and therefore sparingly used method, modern HDX MS is more straightforward, rapid, and routine than in the past. As a result, the breadth of applications of the method has expanded. This Review catalogs applications of HDX MS that have appeared in the literature during the 30 months from January 2012 to June 2014. As penetration of the method into nonacademic sectors where confidentiality is necessary is also at an all-time high, many more applications of this method likely exist that have not been reported in the literature. A synopsis of the recent applications of HDX MS is shown in Figure ​Figure11 where classifications have been made in terms of sector in which the work was performed, geography, and general topic. We elected to categorize the publications in these ways to emphasize that the method is used not only in all sectors but also on nearly every continent. Many different possibilities existed for characterizing the applications by topic, and this was not a perfect task. Some papers belong in multiple topics or could arguably be placed in different categories than we finally decided. An Excel database and an Endnote library of the 234 articles we surveyed, both of which contain the topic groupings, are available from the authors. Figure 1 Synopsis of the published applications of HDX MS from January 2012 to June 2014. (A) A total of two-hundred thirty-four (234) articles were published and classified according to the scheme shown, based on author affiliations. The number of articles in ... Academia is the largest sector contributing to published HDX MS applications (Figure ​(Figure1).1). Approximately 25% of the papers surveyed included multiple sectors, which we have classified as mixed. As mentioned above, such categorization is biased against work that has not been published. Industrial research and research from governmental laboratories must/may remain confidential. Therefore, the results in Figure ​Figure1A1A are to be interpreted with this in mind. The United States was the primary source of publications in HDX MS during the January 2012 to June 2014 time period (Figure ​(Figure1B,C),1B,C), although significant and important work originated from 22 other countries (Figure ​(Figure1D).1D). To illustrate the HDX MS applications in much greater detail, we have divided the remainder of the article into six sections that each discuss one of the topical classifications shown in Figure ​Figure11E.

140 citations