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137


Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

Stanley, Frederick M; Linder, Kathryn M; Cardozo, Timothy J
Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter.
PMCID:4574702
PMID: 26379245
ISSN: 1932-6203
CID: 1779342

Response to letter to the editor [Letter]

Shmelkov, Evgeny; Cardozo, Timothy
PMCID:4514404
PMID: 25759130
ISSN: 2164-554x
CID: 1568562

Quantitative analysis of T cell receptor complex interaction sites using genetically encoded photo-cross-linkers

Wang, Wenjuan; Li, Tianqi; Felsovalyi, Klara; Chen, Chunlai; Cardozo, Timothy; Krogsgaard, Michelle
The T cell receptor (TCR)-cluster of differentiation 3 (CD3) signaling complex plays an important role in initiation of adaptive immune responses, but weak interactions have obstructed delineation of the individual TCR-CD3 subunit interactions during T cell signaling. Here, we demonstrate that unnatural amino acids (UAA) can be used to photo-cross-link subunits of TCR-CD3 on the cell surface. Incorporating UAA in mammalian cells is usually a low efficiency process. In addition, TCR-CD3 is composed of eight subunits and both TCR and CD3 chains are required for expression on the cell surface. Photo-cross-linking of UAAs for studying protein complexes such as TCR-CD3 is challenging due to the difficulty of transfecting and expressing multisubunit protein complexes in cells combined with the low efficiency of UAA incorporation. Here, we demonstrate that by systematic optimization, we can incorporate UAA in TCR-CD3 with high efficiency. Accordingly, the incorporated UAA can be used for site-specific photo-cross-linking experiments to pinpoint protein interaction sites, as well as to confirm interaction sites identified by X-ray crystallography. We systemically compared two different photo-cross-linkers-p-azido-phenylalanine (pAzpa) and H-p-Bz-Phe-OH (pBpa)-for their ability to map protein subunit interactions in the 2B4 TCR. pAzpa was found to have higher cross-linking efficiency, indicating that optimization of the selection of the most optimal cross-linker is important for correct identification of protein-protein interactions. This method is therefore suitable for studying interaction sites of large, dynamic heteromeric protein complexes associated with various cellular membrane systems.
PMCID:4168801
PMID: 25061810
ISSN: 1554-8929
CID: 1252232

Vaccine focusing to cross-subtype HIV-1 gp120 variable loop epitopes

Cardozo, Timothy; Wang, Shixia; Jiang, Xunqing; Kong, Xiang-Peng; Hioe, Catarina; Krachmarov, Chavdar
We designed synthetic, epitope-focused immunogens that preferentially display individual neutralization epitopes targeted by cross-subtype anti-HIV V3 loop neutralizing monoclonal antibodies (mAbs). Vaccination of rabbits with these immunogens resulted in the elicitation of distinct polyclonal serum Abs that exhibit cross-subtype neutralization specificities mimicking the mAbs that guided the design. Our results prove the principle that a predictable range of epitope-specific polyclonal cross-subtype HIV-1 neutralizing Abs can be intentionally elicited in mammals by vaccination. The precise boundaries of the epitopes and conformational flexibility in the presentation of the epitopes in the immunogen appeared to be important for successful elicitation. This work may serve as a starting point for translating the activities of human broadly neutralizing anti-HIV-1 monoclonal antibodies (bNAbs) into matched immunogens that can contribute to an efficacious HIV-1 vaccine.
PMCID:4138239
PMID: 25045827
ISSN: 0264-410x
CID: 1131812

Specific Increase in Potency via Structure-Based Design of a TCR

Malecek, Karolina; Grigoryan, Arsen; Zhong, Shi; Gu, Wei Jun; Johnson, Laura A; Rosenberg, Steven A; Cardozo, Timothy; Krogsgaard, Michelle
Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive "self" proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in approximately 30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag-MHC (peptide-MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.
PMCID:4205480
PMID: 25070852
ISSN: 0022-1767
CID: 1089952

Sequence Conserved and Antibody Accessible Sites in the V1V2 Domain of HIV-1 gp120 Envelope Protein

Shmelkov, Evgeny; Grigoryan, Arsen; Krachmarov, Chavdar; Abagyan, Ruben; Cardozo, Timothy J
The immune-correlates analysis of the RV144 trial suggested that epitopes targeted by protective antibodies (Abs) reside in the V1V2 domain of gp120. We mapped V1V2 positional sequence variation onto the conserved V1V2 structural fold and showed that while most of the solvent accessible V1V2 amino acids vary between strains, there are two accessible molecular surface regions that are conserved and also naturally antigenic. These sites may contain epitopes targeted by broadly cross-reactive anti-V1V2 antibodies.
PMCID:4151074
PMID: 25051095
ISSN: 0889-2229
CID: 1075922

Identification and Characterization of Small Molecules That Inhibit Nonsense-Mediated RNA Decay and Suppress Nonsense p53 Mutations

Martin, Leenus; Grigoryan, Arsen; Wang, Ding; Wang, Jinhua; Breda, Laura; Rivella, Stefano; Cardozo, Timothy; Gardner, Lawrence B
Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTC) and the rapid degradation of their mRNAs by nonsense-mediated RNA decay (NMD). We used virtual library screening, targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD-targeted mRNAs at nanomolar concentrations, with minimal toxicity in cell-based assays. As expected, pharmacologic NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC-mutated p53, pharmacologic NMD inhibition combined with a PTC "read-through" drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacologic NMD inhibitors can restore mRNA integrity in the presence of PTC and can be used as part of a strategy to restore full-length protein in a variety of genetic diseases. Cancer Res; 74(11); 3104-13. (c)2014 AACR.
PMCID:4040335
PMID: 24662918
ISSN: 0008-5472
CID: 1032302

Computational Prediction of Neutralization Epitopes Targeted by Human Anti-V3 HIV Monoclonal Antibodies

Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy
The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.
PMCID:3934971
PMID: 24587168
ISSN: 1932-6203
CID: 829652

Topology Influences V2 Epitope Focusing [Meeting Abstract]

Shmelkov, Sergey; Rao, Mangala; Wang, Shixia; Seaman, Michael; Kong, Xiangpeng; Lu, Shan; Cardozo, Timothy
ISI:000344774402125
ISSN: 1931-8405
CID: 1882932

Could vaccination with AIDSVAX immunogens have resulted in antibody-dependent enhancement of HIV infection in human subjects?

Shmelkov, Evgeny; Nadas, Arthur; Cardozo, Timothy
The immune-correlate analysis of the RV144 clinical trial revealed that human plasma IgA immune responses elicited by the RV144 vaccine correlated positively with a risk for HIV acquisition. This result once again emphasized that HIV vaccines can potentially have adverse effects leading to enhancement of infection. Here, we discuss previously reported evidence of antibody-dependent enhancement of HIV infection. We also describe how a structure-based epitope-specific sieve-analysis can be employed to mine the molecular mechanism underlying this phenomenon.
PMCID:5443089
PMID: 25483466
ISSN: 2164-5515
CID: 1471382