Imaging of intracellular protein aggregates through plasmon-assisted clusteroluminescence.

The formation of clusters in non-aromatic molecules can give rise to unconventional luminescence or clusteroluminescence. Typically containing heteroatoms without extended conjugation or aromatic rings, these molecules have drawn much attention owing to the prospects of label-free biological imaging. However, their applications have been limited due to the lack of knowledge of the underlying mechanism. Herein, we have elucidated the mechanism of clusteroluminescence from proteins, which were explicitly aggregated using plasmonic silver nanoparticles. The nanoparticles promoted protein aggregation and induced nitrile formation on the surface, which, along with other lone-pair-containing heteroatoms, contributed to enhanced emission in the visible range. Remarkably, this makes imaging of proteins possible with visible excitations, as co-factor-lacking proteins generally undergo electronic transitions only in the ultraviolet range. Furthermore, the inherent protein-aggregating behaviour of plasmonic nanoparticles was harnessed for imaging of intracellular Huntingtin protein aggregates overexpressed in HeLa cells through clusteroluminescence. Significant plasmon-enhanced and red-shifted fluorescence emission was observed, which helped in the imaging and localization of the intracellular aggregates. Density functional theory calculations and transient absorbance spectroscopy were used to probe the molecular interactions at the protein-nanoparticle interface and the charge transfer states, further elucidating the role of nanoparticles and the emission mechanism. This technique thus opens alternate avenues for label-free fluorescence bioimaging.

Unravelling HDAC Selectivity for Erasing Acetyl Mark on Lys-5 of Histone H2B.

The reversible acetylation of specific Lysine residues of histones plays crucial role in the epigenetic regulation of chromatin activity. Importantly, perturbations of acetylation-deacetylation dynamics have important implications for cancer and neurological disorders. There are 18 human HDACs including sirtuins. The site-selective acetyl eraser specificity of HDACs is poorly defined. Deciphering the site specificity preference of HDACs from a gamut of lysine in histones may be critical for targeted inhibitor development and delineation of regulatory mechanisms associated with chromatin. Here, we have interrogated the propensity of HDACs to erase acetyl mark at Lys-5 of H2B namely, H2BK5Ac engineered by a peptide ligation reaction catalyzed by transpeptidase sortase. HDACs and Sirtuins were individually over-expressed in HEK293 cells and the deacetylation propensity of respective cell lysates was evaluated against H2BK5Ac for initial screening of potential acetyl erasers. This screen indicated HDAC1 as the prime eraser of acetyl mark in H2BK5Ac. The propensity of HDAC1 to erase acetyl mark of H2BK5Ac was further probed using semisynthetic designer nucleosomes with whole cell lysates, recombinant enzyme, and specific inhibitors. Consistent with the above data, siRNA knockdown of HDAC1 and closely related HDAC3 in HEK293 cells prevented the loss of H2BK5 acetylation.

The LEM-ESCRT toolkit: Repair and maintenance of the nucleus.

The eukaryotic genome is enclosed in a nuclear envelope that protects it from potentially damaging cellular activities and physically segregates transcription and translation.Transport across the NE is highly regulated and occurs primarily via the macromolecular nuclear pore complexes.Loss of nuclear compartmentalization due to defects in NPC function and NE integrity are tied to neurological and ageing disorders like Alzheimer's, viral pathogenesis, immune disorders, and cancer progression.Recent work implicates inner-nuclear membrane proteins of the conserved LEM domain family and the ESCRT machinery in NE reformation during cell division and NE repair upon rupture in migrating cancer cells, and generating seals over defective NPCs. In this review, we discuss the recent in-roads made into defining the molecular mechanisms and biochemical networks engaged by LEM and many other integral inner nuclear membrane proteins to preserve the nuclear barrier.

Unraveling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.

Aurora kinases are critical mitotic serine/threonine kinases and are often implicated in tumorigenesis. Recent studies of the interphase functions for aurora kinase (Aurk)A have considerably expanded our understanding of its role beyond mitosis. To identify the unknown targets of AurkA, we used peptide array-based screening and found E2F4 to be a novel substrate. Phosphorylation of E2F4 by AurkA at Ser75 regulates its DNA binding and subcellular localization. Because E2F4 plays an important role in skeletal muscle differentiation, we attempted to gain insight into E2F4 phosphorylation in this context. We observed that a block in E2F4 phosphorylation retained it better within the nucleus and inhibited muscle differentiation. RNA sequencing analysis revealed a perturbation of the gene network involved in the process of muscle differentiation and mitochondrial biogenesis. Collectively, our findings establish a novel role of AurkA in the process of skeletal muscle differentiation.-Dhanasekaran, K., Bose, A., Rao, V. J., Boopathi, R., Shankar, S. R., Rao, V. K., Swaminathan, A., Vasudevan, M., Taneja, R., Kundu, T. K. Unravelling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.

Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression.

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.

Noncoding RNAs in chromatin organization and transcription regulation: an epigenetic view.

The Genome of a eukaryotic cell harbors genetic material in the form of DNA which carries the hereditary information encoded in their bases. Nucleotide bases of DNA are transcribed into complimentary RNA bases which are further translated into protein, performing defined set of functions. The central dogma of life ensures sequential flow of genetic information among these biopolymers. Noncoding RNAs (ncRNAs) serve as exceptions for this principle as they do not code for any protein. Nevertheless, a major portion of the human transcriptome comprises noncoding RNAs. These RNAs vary in size, as well as they vary in the spatio-temporal distribution. These ncRnAs are functional and are shown to be involved in diverse cellular activities. Precise location and expression of ncRNA is essential for the cellular homeostasis. Failures of these events ultimately results in numerous disease conditions including cancer. The present review lists out the various classes of ncRNAs with a special emphasis on their role in chromatin organization and transcription regulation.

Inhibition of acetyltransferase alters different histone modifications: probed by small molecule inhibitor plumbagin.

Histone modifications; acetylation, methylation (both Lysine and Arginine) etc., at different positions regulates the chromatin fluidity and function in a combinatorial manner, which could be referred as an epigenetic language. In the context of transcription, histone acetylation, methylation and phosphorylation at specific sites, especially at the N-terminal tails of histones play very important roles in activation and/or repression. While acetylation of histones is generally important for transcriptional activation, methylation and phosphorylation could also be involved in repression, depending on the context. Here, we have investigated the crosstalk of histone modifications on a gross scale over histone H3, using a small molecule inhibitor of lysine acetyltransferase KAT3B/p300, Plumbagin, to analyze the histone modification profile upon inhibition of acetylation. In addition to the inhibition of acetylation, there was a concomitant decrease of transcriptional activation mark, H3 lysine 4 trimethylation (H3K4me3) in the cellular context. The histone H3 Serine 10 Phosphorylation (H3S10p) also decreased upon inhibition of acetylation. However, there were no changes observed with transcriptional repressive marks like H3 Lysine 9 di/trimethylation (H3K9me2/me3) suggesting that transcriptional activation marks were selectively targeted. These data suggest that Plumbagin induces a distinct modification profile involving transcriptional activation marks H3K4me3 and H3S10 phosphorylation in the context of histone acetylation brought about by KAT3B/ p300.