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Mortalin, a member of Hsp70 family, was first cloned as a mortality factor in mouse fibroblasts in 1993. It was subsequently determined as a key player of mitochondrial biogenesis, ATP production, stress responses, chaperoning, intracellular trafficking and continued proliferation of cancer cells. Besides its role in cancer, mortalin is also an important protein for the brain that utilizes high energy and depends heavily on mitochondrial functions. In a proteomic screening, mortalin was detected as a downregulated protein in Parkinson’s disease and oxidized in Alzheimer’s disease depicting that the “functional lack of mortalin” is related to these diseases. Knockdown of mortalin homologue in worms (C. elegans) caused abnormalities in mitochondria, premature senescence and progeria like phenotype. These studies have demonstrated that mortalin-mediated mitochondrial functions are not only the key factors in maintaining the continued proliferation of cancer cells but also the normal neuronal physiology. Lack of a functional mortalin leads to cancer cell death and neurodegenerative phenotype.

Normal cells can divide only for a limited number of times in vitro (Hayflick’s limit) and reach an irreversible non-proliferative state referred to as replicative cellular senescence. Since many tumor suppressor genes that are inactivated in cancers are highly expressed in senescing cells, cellular senescence is widely regarded as a tumor suppressor mechanism that acts as a safeguard for cancer. Indeed, forced expression of tumor suppressor proteins have been shown to induce growth arrest of cancer cells that often mimics replicative senescence. Mortalin is a member of the Hsp70 family of proteins and exhibits pancytoplasmic distribution pattern in normal and perinuclear in human cancer cells. Cancer cells when induced to senesce by a variety of chemicals, such as BrdU, 5AZA-dC, MKT-077 and phytochemicals, showed shift in mortalin staining pattern from the perinuclear to the pancytoplasmic type, associated with nuclear translocation and activation of p53 function. Using mortalin staining as a model reporter, human shRNA library was screened for anticancer candidate. Bioinformatics analysis of the gene targets of the selected shRNAs revealed the involvement of DNA damage signaling pathway as a candidate target for anticancer therapy. In this chapter, use of mortalin staining as a reporter for anticancer drug screening and its outcome will be discussed.

Nanocarriers are the small molecules with capability of adhering to or pass through the cell membrane and deliver the genes, peptides, growth factors or toxins to living cells. They form an extremely important part of gene delivery and molecular therapeutics. Besides the viral modes of gene delivery, cationic carrier molecules (e.g., liposome and polymers) that form non-covalent complexes with DNA constitute a major class of nanocarriers. Although relatively less efficient than viral systems, cationic carrier molecules have inherent advantage of flexibility and safety. Their derivatives in conjugation with functional molecules such as, peptides, proteins, growth factors and antibodies have been in focus to generate nanocarriers with low toxicity, high stability, high efficiency and cell-specific targeting features. In this chapter, we discuss the use of cell internalizing antibodies against mortalin protein as nanocarriers. Internalizing anti-mortalin antibodies was employed for (i) internalization of nanoparticles (quantum dots, QD) to generate illuminating cells and (ii) gene delivery. Furthermore, cationic polymer polyethylenimine (PEI) and internalizing anti-mortalin antibody complex was shown to enhance the gene delivery specifically to cancer cells and thus enabling the latter to serve as a novel cancer-targeting nanocarrier.

Mortalin is a member of Hsp70 family that exhibits a tight correlation with carcinogenesis. It has been shown to inactivate tumor suppressor protein p53. The present chapter reviews the history and presents information of mortalin, p53 and how their interaction contributes to carcinogenesis. It is demonstrated that mortalin-p53 interaction that is a cancer-driving event depends on the stress state of cells. The small molecules and short peptides that were able to block this interaction could induce apoptosis in cancer cells that harbour mutant p53 suggesting that mortalin-p53 is a good target for cancer therapy. Understanding of the molecular regulation of mortalin-p53 interaction and their role in highly malignant cells will help designing better strategies for cancer treatment.

Mortalin/mtHsp70 performs a wide array of cellular functions and has been implicated in aging, cancer and neurodegenerative diseases. Similar to other Hsp70s, its ability to chaperone misfolded proteins and bind to a myriad of clients is derived from its N-terminal nucleotide-binding domain (NBD) regulating substrate affinity of the C-terminal substrate-binding domain (SBD) in a nucleotide- and co-chaperone-dependent mechanism. To understand the structural dynamics of its allostery making this relevant to mortalin’s cellular function, this chapter describes key structural features of these two domains as well as provide an appreciation as to possibly how a single amino acid change, Gly to Arg in the SBD that can be viewed so minor, is able to metamorphose from a life-extending species of mortalin (mot-2) into one that induces senescence and even inhibits tumor growth (mot-1).

Visualize the invisible is the most recent challenge of modern biotechnology in which the advancement of molecular and cell imaging has been prioritized. Fluorescence microscopy has become an essential tool to study biological molecules, pathways and events in vitro and in vivo for diagnostics and therapeutics. The field not only involves the development of new generation of molecules that are nontoxic and noninvasive, but also concerns their long-term stability, sensitivity and resolution in visual assays. This chapter reviews the use of quantum dots (small, light-emitting semiconductor nanocrystals) in conjugation with the anti-mortalin cell-internalizing antibody for bioimaging. The conjugate was able to enter the cells and illuminate them. The use of such illuminated cells for long-term tracking and imaging in mouse and rabbit models of cartilage and bone differentiation in vitro and in vivo is described.

The mitochondrion, arising from the historical endosymbiosis during the stressful period of the Great Oxidation Event 2.4 billion years ago, marks the existence of all eukaryotes. Retaining only a handful of genes from its ancestral symbiont and yet performing life-essential tasks, it is heavily dependent on the nucleus and a consortium of stress chaperones that maintain its structural and functional integrity by regulation of transport of the nuclear-encoded proteins, their quality control by chaperoning and proteolysis, and energy-generation as a part of their housekeeping and stress-survival functions. Mortalin, first identified in 1993 from cell fusion studies as a marker of mortal cell phenotype, was characterized as an Hsp70 family stress chaperone based on its sequence homology. Nearly two decades of experimental data have revealed its residence beyond the mitochondrial boundaries, life essential functions in and outside the mitochondria and those that specifically promote carcinogenesis on one hand and neurodegeneration on the other. Aimed to portrait mortalin characteristics, both in structure and function and drive the mortalin biology to drug discovery, this chapter reviews the events leading to its identification and role in old age diseases including cancer along with its possibility of being a therapeutic target.

Mortalin, also known as GRP75/mtHSP70/PBP74, is a member of the hsp70 family of proteins. It is frequently enriched in cancers and shows distinct subcellular distribution in normal and immortal/cancer cells. Mortalin-overexpressing immortalized human cells show enhanced malignant properties, including anchorage-independent growth, formation of tumors in nude mice and chemotaxis. Targeting mortalin with antisense, ribozyme or siRNA specific for mortalin induced tumor cell-specific growth arrest and apoptosis suggesting that the development of various modalities for targeting mortalin may be useful in clinical applications. In this chapter, we discuss the various strategies that have been explored to generate mortalin-targeted cancer gene therapeutics.

Overexpression of mortalin in a wide variety of malignancies is generally associated with highly advanced and aggressive cancers. In the past decade, since its discovery, the classic understanding linking the role of this protein to cancer has relied on: (a) its mitochondrial functions (as the main import protein and mitochondrial house- keeper) consonant to the increased energy demand by cancer cells, and/or (b) its pro- proliferative, cytoplasmic sequestration/degradation of wild-type p53. Recent studies have ascribed new tumorigenic functions for mortalin expanding its roles in signal trans- duction, mitochondrial and extra-mitochondrial chaperonization, and even maintenance of genome stability. Recently, we have proposed a "molecular sponge" hypothesis based on chaperone-drug interactions and observed mortalin-dependent response of tumor cells to some anti-cancer agents. These new paradigms on mortalin biology are expected to yield unique promises in biomedicine

“Ribozymes” are RNA molecules with enzymatic properties [1]. These are naturally occurring moieties that provide fine-tuning of the gene expression cascades (replication, transcription and translation) by catalyzing essential steps such as cleavage and ligation [2]. Hammerhead ribozymes (for resemblance of their two-dimensional structure to a hammerhead) are the smallest ribozymes that are used as molecular scissors in molecular biology and biotechnology to elucidate and eliminate gene functions. The RNA is induced to fold into its active conformation by the binding of metal ions. It forms two domains; the scaffold (“domain 2”) on which the ribozyme is built and the active centre (catalytic domain called “domain I”) of the ribozyme [3, 4]. Over the last two decades, mechanism of action of hammerhead ribozymes describing the requirement of divalent metal ion, definition of the catalytic domains and sequence specificity usually referred to as target site have been largely demonstrated [4–6]. Ribozyme activity in vivo critically depends on its effective level of expression, specificity, intracellular stability, target colocalization and accessibility to the target sites [7]. These technical issues imposed a major difficulty in the use of ribozymes in vivo. Various modifications of the ribozyme expression plasmid have subsequently thus come into play. For example, it was shown that the ribozymes expressed under the control of RNA polymerase III promoter (tRNAVal promoter) were efficiently expressed, highly stable and were exported to the cytoplasm [7]. Such expression improved the ribozyme activity in vivo by many folds. However, efficiently expressed and highly stable ribozymes may still lack action due to their inaccessibility to the target sites that in turn imposed a major hurdle because of the unforeseeable secondary and tertiary RNA structures. Recently, this obstacle was elegantly resolved by modifying the ribozyme expression plasmids. A novel hybrid ribozyme that combined the cleavage activity of hammerhead ribozyme with the unwinding activity of the endogenous RNA helicase was developed. Such RNA helicase based hybrid ribozymes expressed from RNA polymerase III promoter (tRNAVal) were indeed shown to have substrate-unwinding as well as strong cleavage activity [8, 9]. Such hybrid ribozymes can attack most, if not all, sites within mRNA. Thus, it was possible to prepare libraries of hybrid ribozymes with randomized binding arms and to examine their effects after they have been introduced into cells. This novel system has many potential applications such as, elucidation of molecular mechanism of biological phenotypes including aging and cancer by identification of novel genes and their intervention by gene targeting. This chapter is dedicated to the molecularand technical details on the use of hammerhead ribozymes and their improved versions for identification of novel functional genes and their role in biological phenotypes.