Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.     

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.     

Genetic structure and diversity analysis of the primary gene pool of chickpea using SSR markers

Members of the primary gene pool of the chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum and four of C. echinospermum grown in India were investigated using 100 SSR markers to analyze their genetic structure, diversity and relationships. We found considerable diversity, with a mean of 4.8 alleles per locus (ranging from 2 to 11); polymorphic information content ranged from 0.040 to 0.803, with a mean of 0.536. Most of the diversity was confined to the wild species, which had higher values of polymorphic information content, gene diversity and heterozygosity than the cultivated species, suggesting a narrow genetic base for cultivated chickpea. An unrooted neighbor-joining tree, principal coordinate analysis and population structure analysis revealed differentiation between the cultivated accessions and the wild species; three cultivated accessions were in an intermediate position, demonstrating introgression within the cultivated group. Better understanding of the structure, diversity and relationships within and among the members of this primary gene pool will contribute to more efficient identification, conservation and utilization of chickpea germplasm for allele mining, association genetics, mapping and cloning gene(s) and applied breeding to widen the genetic base of this cultivated species, for the development of elite lines with superior yield and improved adaptation to diverse environments.     

CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

ABSTRACT: BACKGROUND: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al..., Nature Medicine 17:822[EN DASH]829 (2011)]. RESULTS AND DISCUSSION: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release by compared to vehicle control. CONCLUSIONS: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.     

Disruption of NMDAR–CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model

Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca²⁺ channels. Building on our discovery of the interaction and regulation of Ca²⁺ channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca²⁺ channel. Remarkably, we also found that this region attenuated Ca²⁺ influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2''s role in excitotoxicity and neuroprotection.     

Inhibition of apoptosis, activation of NKT cell and upregulation of CD40 and CD40L mediated by M. leprae antigen(s) combined with Murabutide and Trat peptide in leprosy patients

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose-capped lipoarabinomannan), may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with antigen containing the two immunomodulators in particulate form (liposomes) led to decrease in percentage of propidium iodide positive cells and T cells expressing Fas–FasL as well as decreased caspase-8/-3 activities in lepromatous patients, thereby inhibiting apoptosis, while converse was true upon stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-x_L in lepromatous patients, leading to the inhibition of apoptosis. It was also observed that same formulation upregulated the expression of CD40 on B cells and monocytes-macrophages and CD40L on T cells of lepromatous leprosy patients. The same liposomal formulation significantly increased the expression of CD1b and CD1d on monocytes-macrophages as well as percentage of NKT cells secreting IFN-γ in lepromatous leprosy patients. Thus, the liposomal formulation of antigen with the immunomodulators in vitro promoted the activation of CD40:CD40L pathways and NKT cell function involved in providing cell-mediated immunity to these patients. The same formulation also caused reversal of T cell anergy by inhibiting apoptosis through decreased expression of death receptors (Fas–FasL) and caspase activities (3 and 8) and increased expression of antiapoptotic protein Bcl-x_L in these patients.     

Functional characterization of mutants in the predicted pore region of the rabbit cardiac muscle Ca(2+) release channel (ryanodine receptor isoform 2).

A highly conserved amino acid sequence, GVRAGGGIGD(4831), which may form part of the Ca(2+) release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was then measured by expression in HEK-293 cells, followed by Ca(2+) photometry, high affinity [(3)H]ryanodine binding, and single-channel recording. All mutants except I4829A and I4829T (corresponding to the I4897T central core disease mutant in RyR1) displayed caffeine-induced Ca(2+) release in HEK-293 cells; only mutants G4826A, I4829V, and G4830A retained high affinity [(3)H]ryanodine binding; and single-channel function was found for all mutants tested, except for G4822A and A4825V. EC(50) values for caffeine-induced Ca(2+) release were increased for G4822A, R4824A, G4826A, G4828A, and D4831A; decreased for V4823A; and unchanged for A4825V, G4827A, I4829V, and G4830A. Ryanodine (10 microm), which did not stimulate Ca(2+) release in wild type (wt), did so in Ala mutants in amino acids 4823-4827. It inhibited the caffeine response in wt and most mutants, but enhanced the amplitude of caffeine-induced Ca(2+) release in mutant G4828A. It also restored caffeine-induced Ca(2+) release in mutants I4829A and I4829T. In single-channel recordings, mutants I4829V and G4830A retained normal conductance, whereas all others had decreased unitary channel conductances ranging from 27 to 540 picosiemens. Single-channel modulation was retained in G4826A, I4829V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4829V, and G4830A, in which divalent metals were preferentially conducted, mutants with loss of modulation had no selectivity of divalent cations over a monovalent cation. Analysis of Gly(4822) to Asp(4831) mutants in RyR2 supports the view that this highly conserved sequence constitutes part of the ion-conducting pore of the Ca(2+) release channel and plays a key role in ryanodine and caffeine binding and activation.     

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.