Biomedical subjects

Renu Wadhwa

Publications and source records attributed to Renu Wadhwa.

At least 19 recordsLinked to original sources

PubMed2006

Three faces of mortalin: a housekeeper, guardian and killer.

Mortalin was first cloned as a mortality factor that existed in the cytoplasmic fractions of normal, but not in immortal, mouse fibroblasts. A decade of efforts have expanded its persona from a house keeper protein involved in mitochondrial import, energy generation and chaperoning of misfolded proteins, to a guardian of stress that has multiple binding partners and to a killer protein that contributes to carcinogenesis on one hand and to old age disorders on the other. Being proved to be an attractive target for cancer therapy, it also warrants attention from the perspectives of management of old age diseases and healthy aging.

Animals↗

PubMed2006

Upregulation of mortalin/mthsp70/Grp75 contributes to human carcinogenesis.

Mortalin, also known as mthsp70/GRP75/PBP74, interacts with the tumor suppressor protein p53 and inactivates its transcriptional activation and apoptotic functions. Here, we examined the level of mortalin expression in a large variety of tumor tissues, tumor-derived and in vitro immortalized human cells. It was elevated in many human tumors, and in all of the tumor-derived and in vitro immortalized cells. In human embryonic fibroblasts immortalized with an expression plasmid for hTERT, the telomerase catalytic subunit, with or without human papillomavirus E6 and E7 genes, we found that subclones with spontaneously increased mortalin expression levels became anchorage-independent and acquired the ability to form tumors in nude mice. Furthermore, overexpression of mortalin was sufficient to increase the malignancy of breast carcinoma cells. The study demonstrates that upregulation of mortalin contributes significantly to tumorigenesis, and thus is a good candidate target for cancer therapy.

Animals↗

PubMed2006

Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: relevance to Parkinson disease.

Functional impairment of mitochondria and proteasomes and increased oxidative damage comprise the main pathological phenotypes of Parkinson disease (PD). Using an unbiased quantitative proteomic approach, we compared nigral mitochondrial proteins of PD patients with those from age-matched controls. 119 of 842 identified proteins displayed significant differences in their relative abundance (increase/decrease) between the two groups. We confirmed that one of these, mortalin (mthsp70/GRP75, a mitochondrial stress protein), is substantially decreased in PD brains as well as in a cellular model of PD. In addition, nine candidate mortalin-binding partners were identified as potential mediators of PD pathology. Manipulations of mortalin level in dopaminergic neurons resulted in significant changes in sensitivity to PD phenotypes via pathways involving mitochondrial and proteasomal function as well as oxidative stress.

Age Factors↗

PubMed2006

The factors that contribute to the long-term expression of siRNA.

RNA interference (RNAi) has become a powerful tool of knocking down a specific gene in vivo. In applications of RNAi, chemically synthesized short interfering RNAs (siRNAs) are commonly used because of their easiness and simplicity. However, siRNAs are cost inhibitive and their effect is transient. DNA which encodes siRNA can overcome these problems. In this study, we compared the stability and expression efficiency of plasmid DNA vector, short and long dumbbell-shaped DNA vectors. We found that the short dumbbell-shaped DNA vector is more stable and results in long-term expression of siRNA.

Genetic Vectors↗

PubMed2006

CARF regulates p19ARF-p53-p21WAF1 senescence pathway by multiple checkpoints.

CARF was first cloned as a novel binding partner of ARF from a yeast-interactive screen. CARF and ARF colocalize in the perinucleolar region and have a collaborative function. In the nucleoplasm, CARF interacts with p53 and enhances its function. We demonstrate that p53 downregulates CARF in a negative feedback regulatory loop and may also involve p53 antagonist HDM2.

Animals↗

PubMed2006

Structural and functional differences between mouse mot-1 and mot-2 proteins that differ in two amino acids.

Chaperone functions mediated by the heat-shock protein (HSP) family constitute a fundamental mechanism that governs the life span of organisms. Here we investigated the chaperone activities of the mitochondrial HSP70 protein, mortalin, which is a heat-uninducible stress protein involved in immortalization and tumorigenesis. There are two mortalin alleles, mot-1 and mot-2, in mouse, encoding two distinct proteins. Whereas an overexpression of mot-1-induced senescence in NIH 3T3 cells, overexpression of mot-2 promoted their malignant properties. Here, we provide evidence that mot-1 possesses very low chaperone activity as compared to mot-2. A "lazy lid" hypothesis is proposed for their differential aging phenotypes.

Alleles↗

PubMed2006

Preincubation with the proteasome inhibitor MG-132 enhances proteasome activity via the Nrf2 transcription factor in aging human skin fibroblasts.

Strategies that lead to the upregulation of the proteasome are known to elicit beneficial consequences to the organism by countering oxidative stress-associated disorders, such as protein conformational diseases, cancer, and aging. Mild treatment with proteasome inhibitors has been previously demonstrated to stimulate proteasome activity and cellular resistance against oxidative injury. However, the mechanism for this action has not been clearly defined. We examined the role of the nuclear factor-E2-related factor 2 (Nrf2) in fibroblasts, a key transactivator of the antioxidant response pathway, in the regulation of the proteasome by its inhibitor MG-132. Here, we demonstrate that the stimulation of the proteasome by low levels of MG-132 can be abrogated by small interfering RNAs (siRNAs) targeted against Nrf2. Consistently, cells that constitutively express Nrf2 exhibit elevated levels of proteasome activities. We further investigate how its beneficial effects, that is, proteasome stimulation, are manifested in young and replicative-senescent cells. Our data underscore that manipulation of Nrf2 by the administration of pharmacologically low levels of proteasome inhibitors may prove to be an alternatively potent strategy for inducing long-term protective effects against oxidative stress.

Cells, Cultured↗

PubMed2006

Quantum dot-based protein imaging and functional significance of two mitochondrial chaperones in cellular senescence and carcinogenesis.

Mortalin/mtHSP70 and HSP60 are heat-shock proteins that reside in multiple subcellular compartments, mitochondria being the dominant compartment. We present here biochemical evidence for their in vivo and in vitro interactions. By the use of quantum dots (powerful tools used for simultaneous imaging of multiple proteins), we visualized minute differences in the subcellular niche of these two proteins in normal and cancer cells. Knockdown of either of these two by shRNA expression plasmids caused growth arrest of osteosarcoma cells. However, interestingly, whereas an overexpression of mortalin extended in vitro life span of normal fibroblasts (TIG-1), overexpression of HSP60 was neutral. We demonstrate the minute differences in subcellular distribution of mortalin and HSP60, their involvement in tumorigenesis, and functional distinction in pathways involved in senescence.

Cell Culture Techniques↗

PubMed2006

Geroprotection by glycerol: insights to its mechanisms and clinical potentials.

Chaperones, particularly the heat-shock proteins, are considered as key players in the maintenance of protein homeostasis and are associated with longevity and cellular immortalization. In this study, we investigated the geroprotective activity of the chemical chaperone glycerol. Glycerol showed significant chaperoning activity in refolding heat-denatured luciferase in vivo and in protecting cells from heat stress-induced cytotoxicity. This was accompanied by decrease in p53, an upregulation of a stress chaperone mortalin/mtHsp70, and an increase in proteasome activity in the presence of oxidative stress.

Aging↗

PubMed2006

On the brotherhood of the mitochondrial chaperones mortalin and heat shock protein 60.

The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular "brotherhood", their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging.

Animals↗

PubMed2005

Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60.

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

Cell Division↗

PubMed2005

Activation of wild type p53 function by its mortalin-binding, cytoplasmically localizing carboxyl terminus peptides.

The Hsp70 family member mortalin (mot-2/mthsp70/GRP75) binds to a carboxyl terminus region of the tumor suppressor protein p53. By in vivo co-immunoprecipitation of mot-2 with p53 and its deletion mutants, we earlier mapped the mot-2-binding site of p53 to its carboxyl terminus 312-352 amino acid residues. In the present study we attempted to disrupt mot-2-p53 interactions by overexpression of short p53 carboxyl-terminal peptides. We report that p53 carboxyl-terminal peptides (amino acid residues 312-390, 312-352, 323-390, and 323-352) localize in the cytoplasm, whereas 312-322, 337-390, 337-352, and 352-390 locate mostly in the nucleus. Most interestingly, the cytoplasmically localizing p53 peptides harboring the residues 323-337 activated the endogenous p53 function by displacing it from p53-mortalin complexes and relocating it to the nucleus. Such activation of p53 function was sufficient to cause growth arrest of human osteosarcoma and breast carcinoma cells.

Animals↗

PubMed2005

Quantum dots in bio-imaging: Revolution by the small.

Visual analysis of biomolecules is an integral avenue of basic and applied biological research. It has been widely carried out by tagging of nucleotides and proteins with traditional fluorophores that are limited in their application by features such as photobleaching, spectral overlaps, and operational difficulties. Quantum dots (QDs) are emerging as a superior alternative and are poised to change the world of bio-imaging and further its applications in basic and applied biology. The interdisciplinary field of nanobiotechnology is experiencing a revolution and QDs as an enabling technology have become a harbinger of this hybrid field. Within a decade, research on QDs has evolved from being a pure science subject to the one with high-end commercial applications.

Animals↗

PubMed2005

Mimotope-hormesis and mortalin/grp75/mthsp70: a new hypothesis on how infectious disease-associated epitope mimicry may explain low cancer burden in developing nations.

It is generally observed that countries with heavy infectious burden show lower cancer incidence as compared to more affluent nations. With the emerging paradigm on microbial heat shock proteins (hsps) as molecular link between infections and autoimmune diseases, we posit a new hypothesis, the "mimotope-hormesis", on the immunologic impact of infections on regional cancer prevention. According to this, assaults of infection during early adulthood could fortify the immune system to evoke more potent defenses against late-onset diseases, such as cancer, via autoimmunity. Interestingly, both experimental and clinical data support the beneficial role of autoimmunity in long-term cancer survivors. We illustrate this by a comprehensive in silico mimotope (epitope mimicry) analysis of human infectious pathogens against mortalin (mthsp70/PB74/GRP75), a type of hsp70 protein involved in control of cell proliferation, immortalization and tumorigenesis.

Communicable Diseases↗

PubMed2005

Chemistry-based RNA technologies: demonstration of usefulness of libraries of ribozymes and short hairpin RNAs (shRNAs).

Mechanism of action of hammerhead ribozymes has been investigated and their intracellular activities have been improved. Based on the improved ribozymes and more recently discovered natural RNAi, we have created libraries of both ribozymes and short hairpin RNAs (shRNAs). The introduction of a library of active ribozymes or shRNAs into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function.

Animals↗

PubMed2004

Imminent approaches towards molecular interventions in ageing.

Ageing is an innate feature of living organisms. Sensational progress in its molecular understanding in the last decade has culminated into a highly complex picture. Emerging from this complexity are the distinctive roles of some of the tumor suppressor pathways including p53 and pRB in maintenance of senescence phenotype, and telomere maintaining pathways in its escape. We discuss here the current scenario of molecular ageing and the use of modern approaches for its intervention in culture system, at least. Many of the tools we describe here are the newly emergent functional RNA tools that are proved to be fruitful in decoding the human genome. These post-genomic technologies will help us in the discovery of gene targets for interventions aiming to improve the quality at later years of life beyond their mere algebraic extension.

Aging↗

PubMed2004

Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation.

We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.

Animals↗

PubMed2004

Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes.

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.

Animals↗