Sreejit Parameswaran

Altered expression of calcineurin, calpain, calpastatin and HMWCaMBP in cardiac cells following ischemia and reperfusion.

A rise in intracellular myocardial Ca(2+) during cardiac ischemia activates calpain (Calpn) thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. Calcineurin (CaN) interacts with Calpn and causes cellular damage eventually leading to cell death. Calpastatin (Calp) and high molecular weight calmodulin-binding protein (HMWCaMBP) (homolog of Calp), inhibit Calpn activity and thus prevent cell death. CaN stimulation can also result in self-repair of damaged cardiomyocytes. The present study attempts to elucidate the expression of these proteins in cells under pre-ischemic condition (control), following ischemia induction and also reperfusion subsequent to ischemia. For the first time, flow cytometric analysis (FACS) has been used for analyzing protein expression concurrently with viability. We induced ischemia and subsequently reperfusion in 80% confluent cultures of neonatal murine cardiomyocytes (NMCC). Viability following induction was assessed with 7-AAD staining and the cells were simultaneously checked for protein expression by FACS. We observed that ischemia induction results in increased expression of CaN, Calp and Calpn. HMWCaMBP expression was reduced in live cells following ischemia which suggests that there is a poor survival outcome of cells expressing HMWCaMBP thereby making it a potential biomarker for such cells. Most live cells following ischemia expressed CaN pointing towards self-repair and favorable survival outcomes.

High Molecular Weight Calmodulin-Binding Protein: 20 Years Onwards—A Potential Therapeutic Calpain Inhibitor

Apoptosis in cardiovascular diseases is considered to be a major reason for heart failure. Caspase-independent apoptosis due to calpains and other proteases occurs due to increase in intracellular Ca2+ levels which act on a feed-forward mechanism. Calpains are Ca2+-activated cysteine proteases present in the cytosol as inactive proenzymes. Calpastatin is most efficient and specific calpain inhibitor present in vivo. Earlier, we had reported the expression of novel high molecular weight calmodulin-binding protein (HMWCaMBP) in human and animal cardiac tissue and in very minute quantities in brains and lungs. HMWCaMBP showed calpastatin activity and was also found to be highly homologous to calpastatin I and calpastatin II. Decreased expression of HMWCaMBP was observed during ischemia as it is susceptible to proteolysis by calpains during ischemia-reperfusion. In normal myocardium, HMWCaMBP may protect its substrate from calpains. However, during an early stage of ischemia/reperfusion due to increased Ca2+ influx, calpain activity often exceeds HMWCaMBP activity. This leads to proteolysis of HMWCaMBP and other protein substrates, resulting in cellular damage. The role of HMWCaMBP in ischemia/reperfusion is yet to be elucidated. The present review summarizes the developments in area of HMWCaMBP from the authors’ laboratory and its potential for therapy.

Therapeutic relevance of the protein phosphatase 2A in cancer

Chromosomal Instability (CIN) is regarded as a unifying feature of heterogeneous tumor populations, driving intratumoral heterogeneity. Polo-Like Kinase 1 (PLK1), a serine-threonine kinase that is often overexpressed across multiple tumor types, is one of the key regulators of CIN and is considered as a potential therapeutic target. However, targeting PLK1 has remained a challenge due to the off-target effects caused by the inhibition of other members of the polo-like family. Here we use synthetic dosage lethality (SDL), where the overexpression of PLK1 is lethal only when another, normally non-lethal, mutation or deletion is present. Rather than directly inhibiting PLK1, we found that inhibition of PP2A causes selective lethality to PLK1-overexpressing breast, pancreatic, ovarian, glioblastoma, and prostate cancer cells. As PP2A is widely regarded as a tumor suppressor, we resorted to gene expression datasets from cancer patients to functionally dissect its therapeutic relevance. We identified two major classes of PP2A subunits that negatively correlated with each other. Interestingly, most mitotic regulators, including PLK1, exhibited SDL interactions with only one class of PP2A subunits (PPP2R1A, PPP2R2D, PPP2R3B, PPP2R5B and PPP2R5D). Validation studies and other functional cell-based assays showed that inhibition of PPP2R5D affects both levels of phospho-Rb as well as sister chromatid cohesion in PLK1-overexpressing cells. Finally, analysis of clinical data revealed that patients with high expression of mitotic regulators and low expression of Class I subunits of PP2A improved survival. Overall, these observations point to a context-dependent role of PP2A that warrants further exploration for therapeutic benefits.

Ischemia and reperfusion induce differential expression of calpastatin and its homologue high molecular weight calmodulin-binding protein in murine cardiomyocytes.

In the heart, calpastatin (Calp) and its homologue high molecular weight calmodulin-binding protein (HMWCaMBP) regulate calpains (Calpn) by inhibition. A rise in intracellular myocardial Ca2+ during cardiac ischemia activates Calpn thereby causing damage to myocardial proteins, which leads to myocyte death and consequently to loss of myocardial structure and function. The present study aims to elucidate expression of Calp and HMWCaMBP with respect to Calpn during induced ischemia and reperfusion in primary murine cardiomyocyte cultures. Ischemia and subsequently reperfusion was induced in ∼80% confluent cultures of neonatal murine cardiomyocytes (NMCC). Flow cytometric analysis (FACS) has been used for analyzing protein expression concurrently with viability. Confocal fluorescent microscopy was used to observe protein localization. We observed that ischemia induces increased expression of Calp, HMWCaMBP and Calpn. Calpn expressing NMCC on co-expressing Calp survived ischemic induction compared to NMCC co-expressing HMWCaMBP. Similarly, living cells expressed Calp in contrast to dead cells which expressed HMWCaMBP following reperfusion. A significant difference in the expression of Calp and its homologue HMWCaMBP was observed in localization studies during ischemia. The current study adds to the existing knowledge that HMWCaMBP could be a putative isoform of Calp. NMCC on co-expressing Calp and Calpn-1 survived ischemic and reperfusion inductions compared to NMCC co-expressing HMWCaMBP and Calpn-1. A significant difference in expression of Calp and HMWCaMBP was observed in localization studies during ischemia.

Enhanced protective immunity derived from dendritic cells with phagocytosis of CD40 ligand transgene-engineered apoptotic tumor cells via increased dendritic cell maturation.

Aims and Background Dendritic cells (DCs) play a pivotal role in regulating CD8+ cytotoxic T-lymphocyte (CTL) responses. Currently, DC vaccines have been used in experimental animal models and clinical trials for evaluation of antitumor immunity. However, their efficacy is limited, warranting the improvement of DC-based cancer vaccines. CD40 ligand (CD40L) stimulates DC activation and maturation via CD40-CD40L interaction. We demonstrated that DCs that had phagocytized apoptotic tumor cells induced antitumor immunity. Methods We generated CD40L-expressing (EG7-CD40L) and the control (EG7-Null) EG7 tumor cells by transfection of EG7 tumor cells with CD40L-expressing adenoviral vector AdVCD40L and the control vector AdVpLpA, respectively. We also generated DC vaccines (DC-EG7/CD40L and the control DC-EG7/Null) using DCs with phagocytosis of irradiated EG7-CD40L and EG7-Null tumor cells, and assessed their phenotype and immunogenicity by flow cytometry and animal studies in C57BL/6 mice. Results We demonstrate that an irradiation of 9000-rad induced Annexin V-expressing cell apoptosis in most (~75%) tumor cells, and provide evidence for phagocytosis of apoptotic tumor cells by flow cytometry and confocal microscopy. The DC-EG7/CD40L cells showed higher expression of DC maturation markers (Iab, CD40, CD80, and CD86) and peptide/major histocompatibility complex I than the control DC-EG7/Null cells. In addition, DC-EG7/CD40L vaccine stimulates more efficient (0.97%) tumor-specific CTL responses than DC-EG7/Null cells (0.31%). Furthermore, 80% (4/5) of mice immunized with DC-EG7/CD40L vaccine become tumor-free after EG7 tumor cell challenge, whereas DC-EG7/Null vaccine only delays immunized mouse death. Conclusions Dendritic cells that have phagocytized CD40L-expressing apoptotic tumor cells appear to offer new strategies in DC cancer vaccines.

An integrated computational and experimental study uncovers FUT9 as a metabolic driver of colorectal cancer

Metabolic alterations play an important role in cancer and yet, few metabolic cancer driver genes are known. Here we perform a combined genomic and metabolic modeling analysis searching for metabolic drivers of colorectal cancer. Our analysis predicts FUT9, which catalyzes the biosynthesis of Ley glycolipids, as a driver of advanced‐stage colon cancer. Experimental testing reveals FUT9s complex dual role; while its knockdown enhances proliferation and migration in monolayers, it suppresses colon cancer cells expansion in tumorspheres and inhibits tumor development in a mouse xenograft models. These results suggest that FUT9s inhibition may attenuate tumor‐initiating cells (TICs) that are known to dominate tumorspheres and early tumor growth, but promote bulk tumor cells. In agreement, we find that FUT9 silencing decreases the expression of the colorectal cancer TIC marker CD44 and the level of the OCT4 transcription factor, which is known to support cancer stemness. Beyond its current application, this work presents a novel genomic and metabolic modeling computational approach that can facilitate the systematic discovery of metabolic driver genes in other types of cancer.

Novel myristoylation of the sperm-specific hexokinase 1 isoform regulates its atypical localization

ABSTRACT The hexokinase 1 variant in mammalian spermatozoa (HK1S) has a unique N-terminus and this isoform atypically localizes to the plasma membrane. However, the mechanism of this process currently remains ambiguous. In this report, we show that fatty acylation underlies the specific sorting of HK1S. Employing chimeric reporter constructs, we first established that compartmentalization of HK1S does not function exclusively in sperm cells and that this feature is swappable to somatic HEK293 cells. Although the N-terminus lacks the classical consensus signature for myristoylation and the sequence-based predictions fail to predict myristoylation of HK1S, complementary experimental approaches confirmed that HK1S is myristoylated. Using live-cell confocal microscopy, we show that the mutation of a single amino acid, the myristoyl recipient Gly2, impedes the prominent feature of plasma membrane association and relocates the enzyme to the cytosol but not the nucleus. Additionally, substitutions of the putatively palmitoylated Cys5 is also reflected in a similar loss of compartmentalization of the protein. Taken together, our findings conclusively demonstrate that the N-terminal ‘MGQICQ’ motif in the unique GCS domain of HK1S acquires hydrophobicity by dual lipidic modifications, N-myristoylation and palmitoylation, to serve the requirements for membranous associations and thus its compartmentalization. Summary: Dual acylation (i.e. myristoylation and palmitoylation) regulates the atypical localization of sperm-specific hexokinase 1 isoform and thus is essential for ‘rewiring’ of glycolytic network.

Expression of calcineurin, calpastatin and heat shock proteins during ischemia and reperfusion

Objective Calcineurin (CaN) interacts with calpains (Calpn) and causes cellular damage eventually leading to cell death. Calpastatin (Calp) is a specific Calpn inhibitor, along with CaN stimulation has been implicated in reduced cell death and self-repair. Molecular chaperones, heat shock proteins (Hsp70 and Hsp90) acts as regulators in Calpn signaling. This study aims to elucidate the role of CaN, Calp and Hsps during induced ischemia and reperfusion in primary cardiomyocyte cultures (murine). Methods and results Protein expression was analyzed concurrently with viability using flow cytometry (FACS) in ischemia- and reperfusion-induced murine cardiomyocyte cultures. The expression of Hsp70 and Hsp90, both being molecular chaperones, increased during ischemia with a concurrent increase in death of cells expressing these proteins. The relative expression of Hsp70 and Hsp90 during ischemia with respect to CaN was enhanced in comparison to Calp. Reperfusion slightly decreased the number of cells expressing these chaperones. There was no increase in death of cells co-expressing Hsp70 and Hsp90 along with CaN and Calp. CaN expression peaked during ischemia and subsequent reperfusion reduced its expression and cell death. Calp expression increased both during ischemia and subsequent reperfusion but cell death decreased during reperfusion. Conclusion The present study adds to the existing knowledge that Hsp70, Hsp90, CaN and Calp interact with each other and play significant role in cardio protection.

Role of Calpains in Calmodulin Regulated Systems

Dysregulation of proteolytic enzymes may disrupt normal biological processes in myocardium can lead to various cardiac conditions. Substantial evidence supports the involvement of matrix metalloproteinase, cystine and serine protease families in this process. Calpain is an intracellular Ca2+-activated protease. Deregulation of calpain caused by a disruption of calcium homeostasis during cardiac pathologies such as atrial fibrillation, heart failure, hypertrophy, or ischemia reperfusion, and thus the myocardial damage. Calpain-calcineurin signalling is pivotal in cardiac conditions especially ischemia, since the signalling produces a cascading effect on the outcome of ischemia. The cleavage of phosphodiesterase1 by calpain is crucial for the regulation of cyclic nucleotides especially cAMP and cGMP. Turnover of cAMP and cGMP in cardiac tissue determines how the cells respond and survive to ischemic insult. Among the known calpain inhibitors, the most specific and potent inhibitor is calpastatin, which belongs to the calpain family. Further research on calpain structures will help in determining the conditions required for activation and the ability of calpain specifically proteolyse even untagged substrates. Though many interesting reviews have covered on the entire calpain system, the current manuscript focuses on the research carried out on calpains in relation to cardiac system by describing their interaction with 2 important cardiac specific proteins—calcineurin and phosphodiesterase 1.

Expression-based analyses indicate a central role for hypoxia in driving tumor plasticity through microenvironment remodeling and chromosomal instability

Can transcriptomic alterations drive the evolution of tumors? We asked if changes in gene expression found in all patients arise earlier in tumor development and can be relevant to tumor progression. Our analyses of non-mutated genes from the non-amplified regions of the genome of 158 triple-negative breast cancer (TNBC) cases identified 219 exclusively expression-altered (EEA) genes that may play important role in TNBC. Phylogenetic analyses of these genes predict a “punctuated burst” of multiple gene upregulation events occurring at early stages of tumor development, followed by minimal subsequent changes later in tumor progression. Remarkably, this punctuated burst of expressional changes is instigated by hypoxia-related molecular events, predominantly in two groups of genes that control chromosomal instability (CIN) and those that remodel tumor microenvironment (TME). We conclude that alterations in the transcriptome are not stochastic and that early-stage hypoxia induces CIN and TME remodeling to permit further tumor evolution.Tumorigenesis: Hypoxia affects CIN and TME genes simultaneouslyAltered gene expression is universal in tumors, but does it impact tumor development and progression? A collaborative team lead by Franco Vizeacoumar and Jei Han from Canada’s University of Saskatchewan and University of Alberta respectively, analyzed expression of non-mutated and non-amplified genes in Triple-Negative Breast Cancer (TNBC) patients. This exclusively expression-altered genes, included those that regulate chromosomal instability (CIN) and remodeling of tumor microenvironment (TME). Further analyses predict hypoxia induced a “punctuated burst” of changes in expression patterns of genes involved in CIN and TME, driving early stages of tumor development. This work provides a novel strategy to co-inhibit CIN and TME genes to disrupt tumor development.