Role of Ubiquitin Protein Ligases in the Pathogenesis of Polyglutamine Diseases

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age related neurodegenerative disorders including polyglutamine diseases. Appearances of aggregates of the misfolded mutant disease proteins suggest that the cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. The quality control ubiquitin ligases are now increasingly implicated in the biology of polyglutamine diseases, Parkinson’s diseases, Amyotrophic lateral sclerosis and Alzheimer’s disease. Here we review the recent studies that have revealed a critical role of E3 ubiquitin ligases in understanding the pathogenesis of polyglutamine diseases.     

S-phase arrest by reactive nitrogen species is bypassed by okadaic acid, an inhibitor of protein phosphatases PP1/PP2A

In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.     

Endorsing cellular competitiveness in aberrant epithelium of oral submucous fibrosis progression: neighbourhood analysis of immunohistochemical attributes

Epithelial abnormality during the transformation of oral submucous fibrosis (OSF) into oral squamous cell carcinoma has been well studied and documented. However, the differential contribution of atrophy and hyperplasia for malignant potentiality of OSF is yet to be resolved. Existing diagnostic conjectures lack precise diagnostic attributes which may be effectively resolved by substantiation of specific molecular pathology signatures. Present study elucidates existence of cellular competitiveness in OSF conditions using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. The concept of field cancerization was contributory in finding correspondence among neighbouring cells of epithelial layers with reference to differential expression of cardinal cancer-related genes [c-Myc (oncogene), p53 (tumour suppressor), and HIF-1α (hypoxia regulator)] which are known to be important sensors in recognizing cellular competitive interface. Our analyses indicate that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighbouring cells which might be responsible to shape the present and future of the pre-malignant condition. Analytical findings indicated association of atrophic epithelium with stress-driven competitive environment having low c-Myc, high-p53, and stable HIF-1α (the looser cells) which undergo apoptosis. Whereas, the cells with high c-Myc⁺ (winner cells) give rise to hyperplastic epithelium via possible mutation in p53. The epithelial dysplasia plausibly occurs due to clonal expansion of c-Myc and p53 positive supercompetitor cells. Present study proposes quantitative IHC along with neighbourhood analysis which might help us to dig deeper on to the interaction among epithelial cell population to provide a better understanding of field cancerization and malignant transformation of pre-malignancy.     

Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification.

A conserved lysine residue in the "P loop" of domain III renders sodium channels highly selective. Conversion of this residue to glutamate, to mimic the homologous position in calcium channels, enables Ca2+ to permeate sodium channels. Because the lysine-to-glutamate mutation converts a positively charged side chain to a negative one, it has been proposed that a positive charge at this position suffices for Na+ selectivity. We tested this idea by converting the critical lysine to cysteine (K1237C) in mu 1 rat skeletal sodium channels expressed in Xenopus oocytes. Selectivity of the mutant channels was then characterized before and after chemical modification to alter side-chain charge. Wild-type channels are highly selective for Na+ over Ca2+ (PCa/PNa < 0.01). The K1237C mutation significantly increases permeability to Ca2+ (PCa/PNa = 0.6) and Sr2+. Analogous mutations in domains I (D400C), II (E755C), and IV (A1529C) did not alter the selectivity for Na+ over Ca2+, nor did any of the domain IV mutations (G1530C, W1531C, and D1532C) that are known to affect monovalent selectivity. Interestingly, the increase in permeability to Ca2+ in K1237C cannot be reversed by simply restoring the positive charge to the side chain by using the sulfhydryl modifying reagent methanethiosulfonate ethylammonium. Single-channel studies confirmed that modified K1237C channels, which exhibit a reduced unitary conductance, remain permeable to Ca2+, with a PCa/PNa of 0.6. We conclude that the chemical identity of the residue at position 1237 is crucial for channel selectivity. Simply rendering the 1237 side chain positive does not suffice to restore selectivity to the channel.     

Hydrophilic Acylated Surface Protein A (HASPA) of <Emphasis Type="Italic">Leishmania donovani</Emphasis>: Expression,Purification and Biophysico-Chemical Characterization

Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a “non-classical” pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils. Studies in mouse macrophage RAW264.7 cell lines indicate that r-LdHASPA enhances reactive oxygen species (ROS) production. Co-immunoprecipitation (IP) studies indicate that r-LdHASPA interacts with certain macrophage proteins which however could not be identified unambiguously. The present study provides key insights into the structural and functional aspects of an important Leishmania protein, HASPA, which we believe could be useful for further research on vaccine/drug development.     

Guanidine hydrochloride and urea-induced unfolding of Brugia malayi hexokinase

Guanidine hydrochloride and urea-induced unfolding of B. malayi hexokinase (BmHk), a tetrameric protein, was examined in detail by using various optical spectroscopic techniques, enzymatic activity measurements, and size-exclusion chromatography. The equilibrium unfolding of BmHk by guanidine hydrochloride (GdmCl) and urea proceeded through stabilization of several unique oligomeric intermediates. In the presence of low concentrations of GdmCl, stabilization of an enzymatically active folded dimer of BmHk was observed. However an enzymatically inactive dimer of BmHk was observed for urea-treated BmHk. This is the first report of an enzymatically active dimer of hexokinase from any human filarial parasite. Furthermore, although complete recovery of the native enzyme was observed on refolding of BmHk samples denatured by use of low concentrations of GdmCl or urea, no recovery of the native enzyme was observed for BmHk samples denatured by use of high concentrations of GdmCl or urea.