BLM helicase-dependent and -independent roles of 53BP1 during replication stress-mediated homologous recombination

Mutations in BLM helicase cause Bloom syndrome, characterized by predisposition to all forms of cancer. We demonstrate that BLM, signal transducer 53BP1, and RAD51 interact during stalled replication. Interactions between the three proteins have functional consequences. Lack of 53BP1 decreases the cell survival and enhanced chromosomal aberration after replication arrest. 53BP1 exhibits both BLM-dependent and -independent anti-recombinogenic functions in human and mouse cells. Both BLM and 53BP1 abrogate endogenous RAD51 foci formation and disrupt RAD51 polymerization. Consequently, loss of BLM and 53BP1 synergistically enhances stress-dependent homologous recombination. These results provide evidence regarding the cooperation between BLM and 53BP1 during maintenance of genomic integrity.     

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.     

Screening of mitochondrial mutations and insertion–deletion polymorphism in gestational diabetes mellitus in the Asian Indian population

In this study we scrutinized the association between the A8344G/A3243G mutations and a 9-bp deletion polymorphism with gestational diabetes mellitus (GDM) in an Asian Indian population. The A3243G mutation in the mitochondrial tRNA^Leu(UUR) causes mitochondrial encephalopathy myopathy, lactic acidosis, and stroke-like episodes (MELAS), while the A8344G mutation in tRNA^Lys causes myoclonus epilepsy with ragged red fibers (MERRF). We screened 140 pregnant women diagnosed with GDM and 140 non-GDM participants for these mutations by PCR-RFLP analysis. Both A3243G and A8344G were associated with GDM (A3243: OR-3.667, 95% CI = 1.001–13.43, p = 0.03; A8344G: OR-11.00, 95% CI = 0.6026–200.8, p = 0.04). Mitochondrial DNA mutations contribute to the development of GDM. Our results conclude that mitochondrial mutations are associated with the GDM women in our population. Thus it is important to screen other mitochondrial mutations in the GDM women.     

Phosphorylation of TET Proteins Is Regulated via O-GlcNAcylation by the O-Linked N-Acetylglucosamine Transferase (OGT)

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.     

Structure,function, and epigenetic regulation of BNIP3: a pathophysiological relevance

BCL-2 [B-cell leukemia/lymphoma 2]/adenovirus E1B 19KD interacting protein 3 (BNIP3) is an atypical BH3 domain only containing member of Bcl2 family of proteins. BNIP3 is known to be involved in various cellular processes depending on the cell type and conditions and also shown to play a role in various disease conditions including myocardial ischemia, autophagy and apoptosis. Though its role in autophagy and its pro-death activity have been reported in various studies, recent findings have shown its contradictory role in the regulation of these cellular processes. The various studies have shown its epigenetic regulation in disease development and progression and also found to be cytoprotective. In this review, we have focused on the structural and functional aspects of BNIP3 in relation to recent advances of its role in autophagy and apoptosis. Also its role of epigenetic regulation of several genes involved in various diseases was also discussed.     

Synthesis,Molecular Properties Prediction and Antimicrobial Activity of Imidazolyl Schiff Bases,Triazoles and Azetidinones

Benzylidenehydrazinyl imidazoles ( 3 ) are prepared from 2‐hydrazinyl imidazoles ( 2 ) on treatment with hydrazine. The imine functionality in 3 is utilized to develop 5′‐aryl‐N‐(4‐aryl‐1H‐imidazol‐2‐yl)‐1H‐1,2,3‐triazol‐1‐amines ( 5 ) by 1,3‐dipolar cycloaddition of diazomethane followed by aromatization with I₂ in DMSO. Compounds 3 are also explored to prepare 4′‐aryl‐1‐(4‐aryl‐1H‐imidazol‐2‐ylamino)‐3‐chloroazetidin‐2‐ones ( 6 ) on treatment with chloroacetyl chloride. The Molinspiration calculations predicted that 3 , 5 and 6 have molecular hydrophobicity, conformational flexibility, good intestinal absorption and bioactivity scores. The chloro, bromo and nitro substituted imidazolyl azetidinones ( 6c , 6d , 6f ) and nitro substituted imidazolyl triazole ( 5f ) exhibited excellent antibacterial activity on B. subtilis, whereas chloro and nitro substituted imidazolyl triazoles ( 5c , 5f ) showed prominent antifungal activity on A. niger.