Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective TH1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ∼75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ∼50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.

Metastasis is the major cause of mortality in prostate cancer patients. Factors such as genetic makeup and race play critical role in the outcome of therapies. This study was conducted to investigate the relevance of in metastatic prostate cancer disease in Caucasian and African-Americans. We employed race-specific prostate cancer models, clinical specimens, clinical data mining, gene-microarray, transcription-reporter assay, chromatin-immunoprecipitation (ChIP), IHC, transgenic-(tgfl/fl) zebrafish, and mouse metastasis models. BMI1 expression was observed to be elevated in metastatic tumors (lymph nodes, lungs, bones, liver) of Caucasian and African-American prostate cancer patients. The comparative analysis of stage III/IV tumors showed an increased BMI1 expression in African-Americans than Caucasians. TCGA and NIH/GEO clinical data corroborated to our findings. We show that expression (i) positively correlates to metastatic () and (ii) negative correlates to tumor suppressor () levels in tumors. The correlation was prominent in African-American tumors. We show that BMI1 regulates the transcriptional activation of , and We show the effect of pharmacological inhibition of BMI1 on the metastatic genome and invasiveness of tumor cells. Next, we show the anti-metastatic efficacy of BMI1-inhibitor in transgenic zebrafish and mouse metastasis models. Docetaxel as monotherapy has poor outcome on the growth of metastatic tumors. BMI1 inhibitor as an adjuvant improved the taxane therapy in race-based and models. BMI1, a major driver of metastasis, represents a promising therapeutic target for treating advanced prostate cancer in patients (including those belonging to high-risk group).

Gene rearrangement is reported to be associated to the aggressive phenotype and poor prognosis in prostate cancer. We identified a gene fusion between a transcription repressor (BMI1) and transcriptional factor (COMMD3) in human prostate cancer. We show that COMMD3:BMI1 fusion expression is significantly increased in prostate cancer disease in an order: normal tissue < primary < metastatic tumors (Mets). Although elevated TMPRSS-ERG/ETV fusion is reported in prostate cancer, we identified a subtype of Mets exhibiting low TMPRSS:ETV and high COMMD3:BMI1. We delineated the mechanism and function of COMMD3 and COMMD3: BMI1 in prostate cancer. We show that COMMD3 level is elevated in prostate cancer cell models, PDX models (adenocarcinoma, NECaP), and Mets. The analysis of TCGA/NIH/ GEO clinical data showed a positive correlation between increased COMMD3 expression to the disease recurrence and poor survival in prostate cancer. We show that COMMD3 drives proliferation of normal cells and promotes migration/ invasiveness of neoplastic cells. We show that COMMD3:BMI1 and COMMD3 regulate C-MYC transcription and C-MYC downstream pathway. The ChIP analysis showed that COMMD3 protein is recruited at the promoter of C-MYC gene. On the basis of these data, we investigated the relevance of COMMD3:BMI1 and COMMD3 as therapeutic targets using in vitro and xenograft mouse models. We show that siRNAmediated targeting of COMMD3:BMI1 and COMMD3 significantly decreases (i) C-MYC expression in BRD/BET inhibitorresistant cells, (ii) proliferation/invasion in vitro, and (iii) growth of prostate cancer cell tumors in mice. The IHC analysis of tumors confirmed the targeting of COMMD3-regulated molecular pathway under in vivo conditions. We conclude that COMMD3:BMI1 and COMMD3 are potential progression biomarkers and therapeutic targets of metastatic prostate cancer.

The Mycobacterium tuberculosis exported repetitive protein (RvErp) is a crucial virulence-associated factor as determined by its role in the survival and multiplication of mycobacteria in cultured macrophages and in vivo. Although attempts have been made to understand the function of Erp protein, its exact role in Mycobacterium pathogenesis is still elusive. One way to determine this is by searching for novel interactions of RvErp. Using a yeast two-hybrid assay, an adenylyl cyclase (AC), Rv2212, was found to interact with RvErp. The interaction between RvErp and Rv2212 is direct and occurs at the endogenous level. The Erp protein of Mycobacterium smegmatis (MSMEG_6405, or MsErp) interacts neither with Rv2212 nor with Ms_4279, the M. smegmatis homologue of Rv2212. Deletion mutants of Rv2212 revealed its adenylyl cyclase domain to be responsible for the interaction. RvErp enhances Rv2212-mediated cyclic AMP (cAMP) production. Also, the biological significance of the interaction between RvErp and Rv2212 was demonstrated by the enhanced survival of M. smegmatis within THP-1 macrophages. Taken together, these studies address a novel mechanism by which Erp executes its function. IMPORTANCE RvErp is one of the important virulence factors of M. tuberculosis. This study describes a novel function of RvErp protein of M. tuberculosis by identifying Rv2212 as its interacting protein.Rv2212 is an adenylyl cyclase (AC) and produces cAMP, one of the prime second messengers that regulate the intracellular survival of mycobacteria. Therefore, the significance of investigating novel interactions of RvErp is paramount in unraveling the mechanisms governing the intracellular survival of mycobacteria. Discerning the molecular mechanisms used by specific mycobacterial proteins involved in infection and virulence requires an understanding of the protein-protein interaction network. The interactions of secretory proteins of Mycobacterium with the host machinery are vital for successful infection. One such secretory protein involved in virulence of Mycobacterium tuberculosis is Erp (Rv3810).The erp gene of M. tuberculosis encodes an ϳ28.0-kDa secretory protein that migrates as a 36.0-kDa protein and is present in all species of mycobacteria. Its disruption results in a marked decrease in virulence, with lower levels of survival not only in in vitro and cell culture assays but also under in vivo conditions (1, 2). It was recently shown that the nature of the erp allele strongly affects the number and the size of the lung lesions in infected animals (3). No homologue of Erp has been found in other bacterial species, making Erp a mycobacterial signature (4).Erp has a composite structure made up of three domains. While the amino-terminal domain (amino acids 1 to 80) and the carboxyterminal domain (amino acids 176 to 284) are conserved, the central domain, consisting of tandem repeats of 5 amino acids based on a PGLTS motif, is subjected to a high level of interspecies variability (1). A signal sequence is present in the amino termi...

◥S100A4 oncoprotein plays a critical role during prostate cancer progression and induces immunosuppression in host tissues. We hypothesized that S100A4-regulated oncogenic activity in immunosuppressed prostate tumors promotes growth of neoplastic cells, which are likely to become aggressive. In the current study, we investigated whether biopsy-S100A4 gene alteration independently predicts the outcome of disease in patients and circulatory-S100A4 is druggable target for treating immunosuppressive prostate cancer. Aided by DECIPHER-genomic test, we show biopsy-S100A4 overexpression as predictive of (i) poor ADT response and (ii) high risk of mortality in 228 radical prostatectomy-treated patients. Furthermore, analysis of tumor genome data of more than 1,000 patients with prostate cancer (PRAD/SU2C/FHCRC studies) validated the association of S100A4-alteration to poor survival and metastasis. We show that increased serum-S100A4 levels are associated to the prostate cancer progression in patients. The prerequisite for metastasis is the escape of tumor cells via vascular system. We show that extracellular-S100A4 protein as a growth factor induces vascular transmigration of prostate cancer cells and bone demineralization thus forms an ideal target for therapies for treating prostate cancer. By employing surface plasmon resonance and isothermal titration calorimetry, we show that mab6B12 antibody interacts with and neutralizes S100A4 protein. When tested for therapeutic efficacy, the mab6B12 therapy reduced the (i) osteoblastic demineralization of bone-derived MSCs, (ii) S100A4target (NFkB/MMP9/VEGF) levels in prostate cancer cells, and (iii) tumor growth in a TRAMPC2 syngeneic mouse model. The immuno-profile analysis showed that mAb6B12-therapyT cells; and (iii) decreased levels of IL5/6/12/13, sTNFR1, and serum-RANTES. We suggest that S100A4-antibody therapy has clinical applicability in treating immunosuppressive prostate cancer in patients.