Low serum PON1 activity: an independent risk factor for coronary artery disease in North-West Indian type 2 diabetics

Posttranslational modifications of proteins have profound effects on many aspects of their function and have received much attention due to the importance of these processes in epigenetic regulation. In this study, we report that deleted azoospermia associated protein 1 (DAZAP1)/proline-rich RNA binding protein (Prrp), a multifunctional RNA binding protein which is essential for spermatogenesis and normal cell growth, is acetylated at Lysine 150 within its RNA binding domain. The acetylation is predominantly observed in nuclear Prrp, and the nonacetylated form is in cytoplasm. Considering that Prrp is a shuttling protein, we suggest that the acetylation cycle at Prrp K150 regulates nucleocytoplasmic transport in cells.     

PI3K-independent AKT activation in cancers: a treasure trove for novel therapeutics

AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors.     

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H_I and H_IV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca²⁺ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca²⁺ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca²⁺ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca²⁺. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.     

Adenine recognition is a key checkpoint in the energy release mechanism of phage T4 DNA packaging motor

ATP is the source of energy for numerous biochemical reactions in all organisms. Tailed bacteriophages use ATP to drive powerful packaging machines that translocate viral DNA into a procapsid and compact it to near-crystalline density. Here we report that a complex network of interactions dictates adenine recognition and ATP hydrolysis in the pentameric phage T4 large "terminase" (gp17) motor. The network includes residues that form hydrogen bonds at the edges of the adenine ring (Q138 and Q143), base-stacking interactions at the plane of the ring (I127 and R140), and cross-talking bonds between adenine, triphosphate, and Walker A P-loop (Y142, Q143, and R140). These interactions are conserved in other translocases such as type I/type III restriction enzymes and SF1/SF2 helicases. Perturbation of any of these interactions, even the loss of a single hydrogen bond, leads to multiple defects in motor functions. Adenine recognition is therefore a key checkpoint that ensures efficient ATP firing only when the fuel molecule is precisely engaged with the motor. This may be a common feature in the energy release mechanism of ATP-driven molecular machines that carry out numerous biomolecular reactions in biological systems.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.     

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis

Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.     

Ack1-mediated androgen receptor phosphorylation modulates radiation resistance in castration-resistant prostate cancer

Androgen deprivation therapy has been the standard of care in prostate cancer due to its effectiveness in initial stages. However, the disease recurs, and this recurrent cancer is referred to as castration-resistant prostate cancer (CRPC). Radiotherapy is the treatment of choice; however, in addition to androgen independence, CRPC is often resistant to radiotherapy, making radioresistant CRPC an incurable disease. The molecular mechanisms by which CRPC cells acquire radioresistance are unclear. Androgen receptor (AR)-tyrosine 267 phosphorylation by Ack1 tyrosine kinase (also known as TNK2) has emerged as an important mechanism of CRPC growth. Here, we demonstrate that pTyr(267)-AR is recruited to the ATM (ataxia telangiectasia mutated) enhancer in an Ack1-dependent manner to up-regulate ATM expression. Mice engineered to express activated Ack1 exhibited a significant increase in pTyr(267)-AR and ATM levels. Furthermore, primary human CRPCs with up-regulated activated Ack1 and pTyr(267)-AR also exhibited significant increase in ATM expression. The Ack1 inhibitor AIM-100 not only inhibited Ack1 activity but also was able to suppress AR Tyr(267) phosphorylation and its recruitment to the ATM enhancer. Notably, AIM-100 suppressed Ack1 mediated ATM expression and mitigated the growth of radioresistant CRPC tumors. Thus, our study uncovers a previously unknown mechanism of radioresistance in CRPC, which can be therapeutically reversed by a new synergistic approach that includes radiotherapy along with the suppression of Ack1/AR/ATM signaling by the Ack1 inhibitor, AIM-100.     

Discovery of liver selective non-steroidal glucocorticoid receptor antagonist as novel antidiabetic agents

Series of benzyl-phenoxybenzyl amino-phenyl acid derivatives (8a-q) are reported as non-steroidal GR antagonist. Compound 8g showed excellent h-GR binding and potent antagonistic activity (in vitro). The lead compound 8g exhibited significant oral antidiabetic and antihyperlipidemic effects (in vivo), along with liver selectivity. These preliminary results confirm discovery of potent and liver selective passive GR antagonist for the treatment of T2DM.