Shairaz Baksh

Resveratrol Inhibits Cardiac Hypertrophy via AMP-activated Protein Kinase and Akt

Whereas studies involving animal models of cardiovascular disease demonstrated that resveratrol is able to inhibit hypertrophic growth, the mechanisms involved have not been elucidated. Because studies in cells other than cardiomyocytes revealed that AMP-activated protein kinase (AMPK) and Akt are affected by resveratrol, we hypothesized that resveratrol prevents cardiac myocyte hypertrophy via these two kinase systems. Herein, we demonstrate that resveratrol reduces phenylephrine-induced protein synthesis and cell growth in rat cardiac myocytes via alterations of intracellular pathways involved in controlling protein synthesis (p70S6 kinase and eukaryotic elongation factor-2). Additionally, we demonstrate that resveratrol negatively regulates the calcineurin-nuclear factor of activated T cells pathway thus modifying a critical component of the transcriptional mechanism involved in pathological cardiac hypertrophy. Our data also indicate that these effects of resveratrol are mediated via AMPK activation and Akt inhibition, and in the case of AMPK, is dependent on the presence of the AMPK kinase, LKB1. Taken together, our data suggest that resveratrol exerts anti-hypertrophic effects by activating AMPK via LKB1 and inhibiting Akt, thus suppressing protein synthesis and gene transcription.

Dynamics of RASSF1A/MOAP-1 Association with Death Receptors†

ABSTRACT RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-α stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

Calreticulin inhibits commitment to adipocyte differentiation.

Calreticulin, an endoplasmic reticulum (ER) resident protein, affects many critical cellular functions, including protein folding and calcium homeostasis. Using embryonic stem cells and 3T3-L1 preadipocytes, we show that calreticulin modulates adipogenesis. We find that calreticulin-deficient cells show increased potency for adipogenesis when compared with wild-type or calreticulin-overexpressing cells. In the highly adipogenic crt−/− cells, the ER lumenal calcium concentration was reduced. Increasing the ER lumenal calcium concentration led to a decrease in adipogenesis. In calreticulin-deficient cells, the calmodulin–Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway was up-regulated, and inhibition of CaMKII reduced adipogenesis. Calreticulin inhibits adipogenesis via a negative feedback mechanism whereby the expression of calreticulin is initially up-regulated by peroxisome proliferator–activated receptor γ (PPARγ). This abundance of calreticulin subsequently negatively regulates the expression of PPARγ, lipoprotein lipase, CCAAT enhancer–binding protein α, and aP2. Thus, calreticulin appears to function as a Ca2+-dependent molecular switch that regulates commitment to adipocyte differentiation by preventing the expression and transcriptional activation of critical proadipogenic transcription factors.

Identification of the Zn2+ binding region in calreticulin.

Calreticulin binds Zn2+ with the relatively high affinity/low capacity. To determine the location of the Zn2+ binding site in calreticulin different domains of the protein were expressed in E. coli, using the glutathione S‐transferase fusion protein system, and their Zn2+‐dependent interaction with Zn2+‐IDA‐agarose were determined. Three distinct domains were used in this study: the N + P‐domain (the first 290 residues); the N‐domain (residues 1–182) and the proline‐rich P‐domain (residues 180–273). The N + P‐domain bound to the Zn2+‐IDA‐agarose and were eluted with an increasing concentration of imidazole. The N‐domain also bound 65Zn2+ as measured by the overlay method. The P‐domain did not interact with the Zn2+‐IDA‐agarose and it did not bind any detectable amount of Zn2+. Chemical modification of calreticulin with diethyl pyrocarbonate indicated that five out of seven histidines were protected in the presence of Zn2+ but they were modified by diethyl pyrocarbonate in the absence of Zn2+ suggesting that these residues may be involved in Zn2+ binding to calreticulin. We conclude that Zn2+ binding sites in calreticulin are localized to the N‐domain of the protein, region that is not involved in Ca2+ binding to calreticulin.

RASSF tumor suppressor gene family: Biological functions and regulation

Genetic changes through allelic loss and nucleic acid or protein modifications are the main contributors to loss of function of tumor suppressor proteins. In particular, epigenetic silencing of genes by promoter hypermethylation is associated with increased tumor severity and poor survival. The RASSF (Ras association domain family) family of proteins consists of 10 members, many of which are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers such as leukemia, melanoma, breast, prostate, neck, lung, brain, colorectal and kidney cancers. In addition to their tumor suppressor function, RASSF proteins act as scaffolding agents in microtubule stability, regulate mitotic cell division, modulate apoptosis, control cell migration and cell adhesion, and modulate NFκB activity and the duration of inflammation. The ubiquitous functions of these proteins highlight their importance in numerous physiological pathways. In this review, we will focus on the biological roles of the RASSF family members and their regulation.

Identification and immunolocalization of calreticulin in pancreatic cells: No evidence for “calciosomes”

In the present study, we have shown that calreticulin is a major Ca(2+)-sequestering protein in pancreatic microsomes. This protein is a peripheral membrane protein and could be extracted from the microsomal membrane with carbonate buffer at pH 11.4. Calreticulin was identified in the membrane fractions by immunoblotting with a specific antibody, by a 45Ca2+ overlay technique, and by NH2-terminal amino acid analysis of the purified protein. Immunocytochemical localization of calreticulin in pancreatic acinar cells and pancreatic fibroblasts showed that the protein is localized to the ER membranes in these cells. We were unable to detect calsequestrin or any calsequestrin-like proteins in the pancreas and found no evidence for the existence of large numbers of specialized, calreticulin-containing vesicles which could be an equivalent of the calsequestrin-containing calciosomes previously reported in this tissue. Purified pancreatic calreticulin binds Ca2+ with both a low and a high capacity (approximately 1 mol of Ca2+/mol of protein and approximately 20-23 mol of Ca2+/mol of protein). The concentrations of Ca2+ required for half-maximal saturation of the low and high capacity sites were approximately 4-6 microM and approximately 1.5 mM, respectively. We conclude that calreticulin, which is confined to the lumen of the ER, plays a major role in Ca2+ storage in pancreatic cells.

RASSF1A: Not a prototypical Ras effector.

The Ras association domain family (RASSF) of genes are commonly silenced by promoter specific methylation in human cancers. After the cloning of the first two family members in early 2000 (RASSF1 and RASSF5), eight other related genes have been identified (RASSF2, 3, 4 and 6–10). The unifying motif amongst all RASSF family members is the presence of the Ras association (RA) domain that could potentially associate with the Ras family of GTPases. Detailed analyses have determined that RASSF family members are tumor suppressor proteins, activators of cell death, cell cycle modulators, microtubule stabilizers and possibly inflammatory mediators linked to NFκB. As such, exploring the biological function of this gene family is needed and if indeed RASSF proteins could be the missing link between Ras signaling and apoptosis. Several RASSF family members have been demonstrated to associate with Ras. However, there is still controversy regarding the ability of RASSF1A to utilize Ras to promote cell death and of the importance of the RASSF1A RA domain. The focus of this review is to highlight the importance of Ras binding to the RASSF family of proteins and discuss what we currently know about the biology of RASSF1A.

TMX1 determines cancer cell metabolism as a thiol-based modulator of ER–mitochondria Ca2+ flux

Cancer cells are critically dependent on ER–mitochondria Ca2+ flux that regulates their bioenergetics. Here, Raturi et al. identify the ER oxidoreductase TMX1 as a thiol-dependent regulator of this intracellular signaling mechanism within cancer cells.

Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance

BackgroundThe PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities.ResultsUsing the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis.ConclusionSince p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities. Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.

Expression and purification of recombinant and native calreticulin

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.