Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins

Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and betaL-crystallin as target proteins, was increased in alphaA(1-172) and decreased in alphaA(1-168) and alphaA(1-162). The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and alphaA(1-172) was nearly the same as that of ADH and alphaA-wt, alphaA(1-168) had lower and alphaA(1-162) had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172) had slightly higher k value than alphaA-wt and alphaA(1-168) and alphaA(1-162) had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172) was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168) was similar to that of alphaA-wt and alphaA(1-162) had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble

hElp3 Directly Modulates the Expression of HSP70 Gene in HeLa Cells via HAT Activity

Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa cells. Finally, complementation and ChIP Assay in yeast showed that hElp3 can not only complement the growth and slow activation of HSP70 (SSA3) gene transcription, but also directly regulates the transcription of SSA3. On the contrary, these functions are lost when the HAT domain is deleted from hElp3. These data suggest that hElp3 can regulate the transcription of HSP70 gene, and the HAT domain of hElp3 is essential for this function. These findings now provide novel insights and evidence of the functions of hELP3 in human cells

Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes

Genomic and Epigenomic Responses to Chronic Stress Involve miRNA-Mediated Programming

Stress represents a critical influence on motor system function and has been shown to impair movement performance. We hypothesized that stress-induced motor impairments are due to brain-specific changes in miRNA and protein-encoding gene expression. Here we show a causal link between stress-induced motor impairment and associated genetic and epigenetic responses in relevant central motor areas in a rat model. Exposure to two weeks of mild restraint stress altered the expression of 39 genes and nine miRNAs in the cerebellum. In line with persistent behavioural impairments, some changes in gene and miRNA expression were resistant to recovery from stress. Interestingly, stress up-regulated the expression of Adipoq and prolactin receptor mRNAs in the cerebellum. Stress also altered the expression of Prlr, miR-186, and miR-709 in hippocampus and prefrontal cortex. In addition, our findings demonstrate that miR-186 targets the gene Eps15. Furthermore, we found an age-dependent increase in EphrinB3 and GabaA4 receptors. These data show that even mild stress results in substantial genomic and epigenomic changes involving miRNA expression and associated gene targets in the motor system. These findings suggest a central role of miRNA-regulated gene expression in the stress response and in associated neurological function

The use or generation of biomedical data and existing medicines to discover and establish new treatments for patients with rare diseases - recommendations of the IRDiRC Data Mining and Repurposing Task Force

The number of available therapies for rare diseases remains low, as fewer than 6% of rare diseases have an approved treatment option. The International Rare Diseases Research Consortium (IRDiRC) set up the multi-stakeholder Data Mining and Repurposing (DMR) Task Force to examine the potential of applying biomedical data mining strategies to identify new opportunities to use existing pharmaceutical compounds in new ways and to accelerate the pace of drug development for rare disease patients. In reviewing past successes of data mining for drug repurposing, and planning for future biomedical research capacity, the DMR Task Force identified four strategic infrastructure investment areas to focus on in order to accelerate rare disease research productivity and drug development: (1) improving the capture and sharing of self-reported patient data, (2) better integration of existing research data, (3) increasing experimental testing capacity, and (4) sharing of rare disease research and development expertise. Additionally, the DMR Task Force also recommended a number of strategies to increase data mining and repurposing opportunities for rare diseases research as well as the development of individualized and precision medicine strategies.Molecular Technology and Informatics for Personalised Medicine and Healt