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Biomedical subjects

Gopalakrishnapillai Anilkumar

Publications and source records attributed to Gopalakrishnapillai Anilkumar.

9 recordsLinked to original sources
PubMed2006

Association of prostate-specific membrane antigen with caveolin-1 and its caveolae-dependent internalization in microvascular endothelial cells: implications for targeting to tumor vasculature.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein with a highly restricted profile of expression. Expression is primarily limited to secretory cells of the prostatic epithelium, with elevated levels observed in prostate cancer. As an integral membrane protein correlated with prostate cancer, PSMA offers a potentially valuable target for immunotherapy. PSMA is also detected in the neovasculature of a variety of solid tumors but not in the endothelial cells of preexisting blood vessels. Although the significance of PSMA expression in these cells remains elusive, this pattern of expression implies that PSMA may perform a functional role in angiogenesis and may offer a therapeutic target for the treatment of a broad spectrum of solid tumors. In this study, we have expressed PSMA in human microvascular endothelial cells and demonstrate that PSMA binds to caveolin-1 and undergoes internalization via a caveolae-dependent mechanism. The association between PSMA and caveolae in endothelial cells may provide important insight into PSMA function and ways to best exploit this protein for therapeutic benefit.

Antigens, Surface↗
PubMed2006

Functional characterization of AAA family FtsH protease of Mycobacterium tuberculosis.

FtsH is a membrane-bound ATP-dependent zinc-metalloprotease which proteolytically regulates the levels of specific membrane and cytoplasmic proteins that participate in diverse cellular functions, and which therefore might be of critical importance to a human pathogen such as Mycobacterium tuberculosis. As the substrates of MtFtsH in mycobacteria are not known, we examined whether recombinant MtFtsH could complement the lethality of a DeltaftsH3::kan mutation in Escherichia coli and elicit proteolytic activity against the known substrates of E. coli FtsH, namely heat shock transcription factor sigma(32) protein, protein translocation subunit SecY and bacteriophage lambdaCII repressor protein. The MtFtsH protein could not only efficiently complement lethality of DeltaftsH3::kan mutation in E. coli, but could also degrade all three heterologous substrates with specificity when expressed in ftsH-null cells of E. coli. These observations probably reveal the degree of conservation in the mechanisms of substrate recognition and cellular processes involving FtsH protease of M. tuberculosis and E. coli.

ATP-Dependent Proteases↗
PubMed2005

Is prostate-specific membrane antigen a multifunctional protein?

Prostate-specific membrane antigen (PSMA) is a metallopeptidase expressed predominantly in prostate cancer (PCa) cells. PSMA is considered a biomarker for PCa and is under intense investigation for use as an imaging and therapeutic target. Although the clinical utility of PSMA in the detection and treatment of PCa is evident and is being pursued, very little is known about its basic biological function in PCa cells. The purpose of this review is to highlight the possibility that PSMA might be a multifunctional protein. We suggest that PSMA may function as a receptor internalizing a putative ligand, an enzyme playing a role in nutrient uptake, and a peptidase involved in signal transduction in prostate epithelial cells. Insights into the possible functions of PSMA should improve the diagnostic and therapeutic values of this clinically important molecule.

Antigens, Neoplasm↗
PubMed2005

N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy.

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.

Animals↗
PubMed2004

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility.

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.

Actins↗
PubMed2004

Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2.

The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2.3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family Zn(2+)-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane of E. coli and M. smegmatis cells respectively. Expression of MsFtsH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The MsftsH gene did not complement lethality of a DeltaftsH3 : : kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely sigma(32) protein and the protein translocase subunit SecY, of E. coli cells.

ATP-Dependent Proteases↗
PubMed2003

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

Adaptor Protein Complex 2↗
PubMed2003

Prostate-specific membrane antigen association with filamin A modulates its internalization and NAALADase activity.

Prostate-specific membrane antigen (PMSA) is an integral membrane protein highly expressed by prostate cancer cells. We reported previously that PSMA undergoes internalization via clathrin-coated pits (Liu et al., Cancer Res., 58: 4055-4060, 1998). In this study we demonstrate that filamin A, an actin cross-linking protein, associates with the cytoplasmic tail of PSMA and that this association of PSMA with filamin is involved in its localization to the recycling endosomal compartment. By ectopically expressing PSMA in filamin-negative and -positive cell lines, we additionally show that filamin binding to PSMA reduces the internalization rate of PSMA and its N-acelylated-alpha linked-acidic dipeptidase activity. These results suggest that filamin might be an important regulator of PSMA function.

Amino Acid Sequence↗