AAV‐mediated expression of BAG1 and ROCK2‐shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury

A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno‐associated virus (AAV)‐mediated over‐expression of BAG1 and ROCK2‐shRNA in the red nucleus to trace [by co‐expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th₈ in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV‐mediated protein over‐expression versus AAV.shRNA‐mediated down‐regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2‐shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

     

Activation of inflammatory response and apoptosis of polymorphonuclear leukocytes in patients with argemone oil poisoning

In the present study, the role of ROS and RNS in activation of inflammatory response and associated molecular events during apoptosis of polymorphonuclear leucocytes (PMNs) in patients from an outbreak of argemone oil (AO) poisoning leading to epidemic dropsy in Lucknow, India was undertaken. It was observed that generation of superoxide radical, nitrite formation and phagocytosis (103–429%) were significantly increased in PMNs of dropsy patients. Furthermore, activities of superoxide dismutase and glutathione peroxidase (GPx) (47–79%) were found to be increased while that of catalase and glutathione reductase (GR) (56–57%) were decreased. Lipid and protein oxidation, nitrotyrosine formation and 8-hydroxydeoxyguanosine (8-OHdG) excretion were significantly enhanced with concomitant depletion of GSH levels (67%) in dropsy patients. In addition, significant elevation of IL-6, IL-8 and TNF-α (68–406%) in plasma was observed. Apoptosis was enhanced (1.5 folds) with increased (2.0–3.6 folds) caspases 3, 8 and 9 activities along with DNA fragmentation (119%). The results suggest that generation of ROS and RNS along with enhancement of secretion of inflammatory mediators leading to DNA damage followed by apoptosis may have an effect on immune system, which in turn may be responsible for histopathological changes in target organs of dropsy patients.     

Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1

The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehyde-binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1(D88A)) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1(C91S)), but not Otub1(D88A), bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1(D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.     

Ribosomal protein L11 recruits miR-24/miRISC to repress c-Myc expression in response to ribosomal stress

c-Myc promotes cell growth by enhancing ribosomal biogenesis and translation. Deregulated expression of c-Myc and aberrant ribosomal biogenesis and translation contribute to tumorigenesis. Thus, a fine coordination between c-Myc and ribosomal biogenesis is vital for normal cell homeostasis. Here, we show that ribosomal protein L11 regulates c-myc mRNA turnover. L11 binds to c-myc mRNA at its 3' untranslated region (3'-UTR), the core component of microRNA-induced silencing complex (miRISC) argonaute 2 (Ago2), as well as miR-24, leading to c-myc mRNA reduction. Knockdown of L11 drastically increases the levels and stability of c-myc mRNA. Ablation of Ago2 abrogated the L11-mediated reduction of c-myc mRNA, whereas knockdown of L11 rescued miR-24-mediated c-myc mRNA decay. Interestingly, treatment of cells with the ribosomal stress-inducing agent actinomycin D or 5-fluorouracil significantly decreased the c-myc mRNA levels in an L11- and Ago2-dependent manner. Both treatments enhanced the association of L11 with Ago2, miR-24, and c-myc mRNA. We further show that ribosome-free L11 binds to c-myc mRNA in the cytoplasm and that this binding is enhanced by actinomycin D treatment. Together, our results identify a novel regulatory paradigm wherein L11 plays a critical role in controlling c-myc mRNA turnover via recruiting miRISC in response to ribosomal stress.     

Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.     

HIV Induces TRAIL Sensitivity in Hepatocytes

Background

HIV infected patients have an increased susceptibility to liver disease due to Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), alcoholic, and non-alcoholic steatohepatitis. Clinically, this results in limited options for antiretroviral therapy and accelerated rates of liver disease, causing liver disease to be the second leading cause of death for HIV infected patients. The mechanisms causing this propensity for liver dysfunction during HIV remains unknown.

Methodology/Principal Findings

We demonstrate that HIV and/or the HIV glycoprotein gp120 ligation of CXCR4 on hepatocytes selectively up-regulates TRAIL R2 expression and confers an acquired sensitivity to TRAIL mediated apoptosis which is mediated by JNK II, but not p38 nor G-proteins.

Conclusions/Significance

These findings suggest that HIV infection renders hepatocytes more susceptible to liver injury during disease states associated with enhanced TRAIL production such as HBV, HCV, or steatohepatitis.     

TRAIL dependent fratricidal killing of gp120 primed hepatocytes by HCV core expressing hepatocytes

The mechanism by which HIV and HCV cooperatively accelerate hepatocyte damage is not clearly understood; however, each virus affects the TRAIL: TRAIL-receptor system. We, therefore, questioned whether the independent effects of HCV and HIV combine to synergistically result in TRAIL dependent hepatocyte killing. We describe that Huh7 hepatocytes treated with HIV gp120 results in both increase TRAIL-R2 expression and an acquired sensitivity to TRAIL mediated killing. Moreover HCV infection and HCV core expression alone in Huh7 cells upregulates TRAIL. Co-incubation of HIV gp120 primed hepatocytes with HCV core expressing hepatocytes results in the selective death of the HIV gp120 primed hepatocytes that is selectively blocked by TRAIL-R2-Fc fusion protein. Liver biopsies from HIV mono-infected patients have increased TRAIL-R2; biopsies from HCV infected patients have increased TRAIL, while co-infected liver biopsies have increased PARP cleavage within hepatocytes indicating enhanced apoptosis. These findings suggest a pathogenic model to understand why HIV/HCV co-infection accelerates liver injury.