Ubiquitin-proteasome system regulation of a key gene regulatory factor, Paf1C.

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3'-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans. The abundances of these components regulate the assembly of Paf1C and/or its functions, thus implying the mechanisms involved in regulating the abundances of the Paf1C components in altered gene expression and hence cellular pathologies. Towards finding the mechanisms associated with the abundances of the Paf1C components, we analyzed here whether the Paf1C components are regulated via targeted ubiquitylation and 26S proteasomal degradation. We find that the Paf1C components except Paf1 do not undergo the 26S proteasomal degradation in both yeast and humans. Paf1 is found to be regulated by the ubiquitin-proteasome system (UPS) in yeast and humans. Alteration of such regulation changes Paf1's abundance, leading to aberrant gene expression. Intriguingly, while the Rtf1 component of Paf1C does not undergo the 26S proteasomal degradation, it is found to be ubiquitylated, suggesting that Rtf1 ubiquitylation could be engaged in Paf1C assembly and/or functions. Collectively, our results reveal distinct UPS regulation of the Paf1C components, Paf1 and Rtf1, in a proteolysis-dependent and -independent manners, respectively, with functional implications.

UPS writes a new saga of SAGA.

SAGA (Spt-Ada-Gcn5-Acetyltransferase), an evolutionarily conserved transcriptional co-activator among eukaryotes, is a large multi-subunit protein complex with two distinct enzymatic activities, namely HAT (Histone acetyltransferase) and DUB (De-ubiquitinase), and is targeted to the promoter by the gene-specific activator proteins for histone covalent modifications and PIC (Pre-initiation complex) formation in enhancing transcription (or gene activation). Targeting of SAGA to the gene promoter is further facilitated by the 19S RP (Regulatory particle) of the 26S proteasome (that is involved in targeted degradation of protein via ubiquitylation) in a proteolysis-independent manner. Moreover, SAGA is also recently found to be regulated by the 26S proteasome in a proteolysis-dependent manner via the ubiquitylation of its Sgf73/ataxin-7 component that is required for SAGA's integrity and DUB activity (and hence transcription), and is linked to various diseases including neurodegenerative disorders and cancer. Thus, SAGA itself and its targeting to the active gene are regulated by the UPS (Ubiquitin-proteasome system) with implications in diseases.

Chromatin and non-chromatin immunoprecipitations to capture protein-protein and protein-nucleic acid interactions in living cells.

Proteins are expressed from genes via sequential biological processes of transcription, mRNA processing, export and translation, and play their roles in maintaining cellular functions via interactions with proteins, DNAs or RNAs. Thus, it is important to study the protein interactions during biological processes in living cells towards understanding their mechanisms-of-action in real time. Methodologies have been developed over the years to study protein interactions in vivo. One state-of-the-art approach is formaldehyde crosslinking-based immuno- or chemi-precipitation to analyze selective as well as genome/proteome-wide interactions in living cells. It is a popular and widely used methodology for cellular analysis of the protein-protein and protein-nucleic acid interactions. Here, we describe this approach to analyze protein-protein/nucleic acid interactions in vivo.

A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription.

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.

CCN6 influences transcription and controls mitochondrial mass and muscle organization.

Mutations in Cellular Communication Network Factor 6 (CCN6) are linked to the debilitating musculoskeletal disease Progressive Pseudo Rheumatoid Dysplasia (PPRD), which disrupts mobility. Yet, much remains unknown about CCN6 function at the molecular level. In this study, we revealed a new function of CCN6 in transcriptional regulation. We demonstrated that CCN6 localizes to chromatin and associates with RNA Polymerase II in human chondrocyte lines. Using zebrafish as a model organism we validated the nuclear presence of CCN6 and its association with RNA Polymerase II in different developmental stages from 10 hpf embryo to adult fish muscle. In concurrence with these findings, we confirmed the requirement of CCN6 in the transcription of several genes encoding mitochondrial electron transport complex proteins in the zebrafish, both in the embryonic stages and in the adult muscle. Reduction in the expression of these genes upon morpholino-mediated knockdown of CCN6 protein expression led to reduced mitochondrial mass, which correlated with defective myotome organization during zebrafish muscle development. Overall, this study suggests that the developmental musculoskeletal abnormalities linked with PPRD could be contributed at least partly by impaired expression of genes encoding mitochondrial electron transport complexes due to defects in CCN6 associated transcriptional regulation.

Leveraging spreadsheet analysis tool for electrically actuated start-up flow of non-Newtonian fluid in small-scale systems.

In this article, we demonstrate the solution methodology of start-up electrokinetic flow of non-Newtonian fluids in a microfluidic channel having square cross-section using Spreadsheet analysis tool. In order to incorporate the rheology of the non-Newtonian fluids, we take into consideration the Ostwald-de Waele power law model. By making a comprehensive discussion on the implementation details of the discretized form of the transport equations in Spreadsheet analysis tool, and establishing the analytical solution for a special case of the start-up flow, we compare the results both during initial transience as well as in case of steady-state scenario. Also, to substantiate the efficacy of the proposed spreadsheet analysis in addressing the detailed flow physics of rheological fluids, we verify the results for several cases with the corresponding numerical results. It is found that the solution obtained from the Spreadsheet analysis is in good agreement with the numerical results-a finding supporting spreadsheet analysis's suitability for capturing the fine details of microscale flows. We strongly believe that our analysis study will open up a new research scope in simulating microscale transport process of non-Newtonian fluids in the framework of cost-effective and non-time consuming manner.

Advancements in Therapeutics via 3D Printed Multifunctional Architectures from Dispersed 2D Nanomaterial Inks.

2D nanomaterials (2DNMs) possess fascinating properties and are found in multifarious devices and applications including energy storage devices, new generation of battery technologies, sensor devices, and more recently in biomedical applications. Their use in biomedical applications such as tissue engineering, photothermal therapy, neural regeneration, and drug delivery has opened new horizons in treatment of age-old ailments. It is also a rapidly developing area of advanced research. A new approach of integrating 3D printing (3DP), a layer-by-layer deposition technique for building structures, along with 2DNM multifunctional inks, has gained considerable attention in recent times, especially in biomedical applications. With the ever-growing demand in healthcare industry for novel, efficient, and rapid technologies for therapeutic treatment methods, 3DP structures of 2DNMs provide vast scope for evolution of a new generation of biomedical devices. Recent advances in 3DP structures of dispersed 2DNM inks with established high-performance biomedical properties are focused on. The advantages of their 3D structures, the sustainable formulation methods of such inks, and their feasible printing methods are also covered. Subsequently, it deals with the therapeutic applications of some already researched 3DP structures of 2DNMs and concludes with highlighting the challenges as well as the future directions of research in this area.

Facile fabrication of electrospun regenerated cellulose nanofiber scaffold for potential bone-tissue engineering application.

In this study, cellulose acetate (CA) solutions (9-15% w/v) prepared in acetone-water (80:20 & 90:10 v/v) system were subjected to electrospinning for fabricating non-woven nanofibrous CA scaffolds (CAS) with average fiber diameters from 300 to 600 nm. Further, regenerated cellulose scaffold (RCS) was obtained by deacetylation of electrospun CAS in alkaline media for varying time periods to find the ideal time required for complete deacetylation. Following deacetylation, RCS was subjected to varying temperatures (60 °C, 80 °C) to observe the possible positive effect of heat treatment on the improvement of mechanical strength. The RCS was characterized using ATR FTIR, SEM for studying its surface chemistry and morphology along with other physio-chemical characterizations such as micro-tensile strength, swelling property, porosity, degradation rate in acidic conditions. The results were analyzed and co-related with variation of composition in solvent system, deacetylation time and heat treatment temperatures to determine the optimal fabricating conditions for RCS. In vitro studies using MC3T3-E1 osteoblast cells were also conducted on the selected RCS samples to evaluate cell adhesion and cell proliferation using SEM and MTT assay analysis. The primary results indicate positive outcome regarding the viability of RCS as potential biomaterial for bone-tissue engineering.