Endoplasmic Reticulum Stress Induces a Caspase-dependent N-terminal Cleavage of RBX1 Protein in B Cells

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.     

Angiotensin I [Phe8-His9] hydrolase and bradykininase from human lung.

Atypical angiotensin I “converting enzyme” (angiotensin I [Phe⁸-His⁹] hydrolase or APHH) was purified from human lung tissue. Two enzyme preparations from different lungs were found to fragment and inactive bradykinin. Fragmentation was demonstrated by electrophoretic techniques and biological inactivation was demonstrated by bioassay. Bradykininase activity was inhibited by low concentrations of 2,3-dimercaptol-propanol (BAL) and ethylenediaminetetraacetic acid (EDTA), but not by phenylmethylsulfonyl fluoride (PMSF) or p-chloromercuriphenyl sulfonic acid (CMPSA). Conversion of angiotensin I was inhibited by BAL, PMSF, and CMPSA but not by EDTA. APHH from a third lung preparation was free of significant bradykininase activity as determined by bioassay. It is concluded that these two enzymatic activities are probably associated with separate enzymes.     

The gene encoding vasoactive intestinal peptide is located on human chromosome 6p21→6qter

Summary Vasoactive intestinal peptide (VIP) is a regulatory neuropeptide involved in a wide variety of functions, among them vasodilation, smooth muscle relaxation, sweat secretion, gastrointestinal peristalsis, and pancreatic function. A deficient VIP-innervation of sweat glands was recently described as a possible pathogenic factor in sweating of cystic fibrosis (CF) patients. To investigate a possible role for a defective VIP-gene in cystic fibrosis, we have used a panel of rodent-human hybrid cells, retaining defined complements of human chromosomes to localize the VIP-gene to the human chromosome region 6p216qter. As the CF gene was recently mapped to chromosome 7, we conclude that the VIP-gene is not the primary gene defect in this disease.     

Evolution of fringing reefs: space and time constraints from the Gulf of Aqaba

This study documents the pattern and rate of reef growth during the late Holocene as revealed by unique geological conditions at the subsiding NW Gulf of Aqaba. We discovered that the modern fringing reef near the city Elat grows on top of a fossil submerged mid-Holocene reef platform. Four coral cores from the fossil platform were dated using the radiocarbon and U-Th methods. The fossil corals range from 5.6±0.1 to 2.4±0.03 ka, constraining the initiation of the modern reef to 2,400 years ago at most. We documented the detailed morphology of the reef using aerial photographs and scuba diving. The survey shows that at its northern end, growth of the 2-km-long reef is inhibited by an active alluvial fan, and it is composed of isolated knolls that are just approaching the sea surface. Towards the south, the knolls are progressively larger and closer together, until they form a continuous reef platform. Along this north-to-south trend we follow the evolution of reef morphology, changes in coral distribution, and the development of a lagoon separated from the open sea. Based on these observations, we suggest a four-stage reef growth model: (a) the reef initiates as coral colonies, forms knolls, and begins to grow upward, limited by the sea surface. (b) Upon reaching the surface, the knolls spread laterally, preferentially parallel to the dominant wave direction assuming an elongated morphology. (c) Continued growth results in adjacent knolls eventually coalescing to form a continuous jagged reef. We interpret the spurs-and-grooves morphology that can be traced across the reef at Elat as remnants of the original trends of knolls. (d) While reef expansion continues, the original knoll trends may be obscured as a massive reef front takes shape. Considering reef growth rates and observations from the modern reef at Elat, this evolution scheme predicts an age range of 10³ years for corals on the reef platform. The range and distribution of radiometric ages we obtained from the fossil reef platform underlying the living Elat reef confirm this hypothesis.     

Seasonality in fish larval assemblage structure within marine reef National Parks in coastal Kenya

Variability of larval assemblages within marine habitats has an effect on population structure of adults, and on patchiness of populations in space and time. However, despite this effect, few studies have examined this variability in the Western Indian Ocean (WIO) especially in relation to marine park boundaries. In this study, we tested the hypothesis that seasonality and biophysical variables influence fish larval assemblage structure within two of the oldest marine parks in Kenya of differing reef structure. Monthly samples were obtained from the parks using oblique plankton tows for 14–24 months. A total of 38 families and 52 genera of fish larvae were obtained from Malindi Marine Park, while 20 genera in 20 families were sampled from the more enclosed Watamu Marine Park but with a connection to an estuarine system. The preflexion larvae comprised over 80% of the total larvae in the parks with the dominant groups being; Gobiidae, Blenniidae, Engraulidae and Apogonidae. The availability of a wide size range of larvae for some groups like Blenniidae, Gobiidae, Engraulidae, Labridae and Scaridae indicated the possibility of some larvae completing their pelagic phase within the parks. Larval densities differed significantly between seasons and stations in both parks, with higher densities ranging between 95–311 larvae/100 m³ during the calm northeast monsoon (NEM) season, compared to 5–58 larvae/100 m³ during the stormy southeast monsoon (SEM) season. Stepwise multiple regression analysis indicated a significant positive influence of zooplankton density on fish larval abundance in Malindi Park, while a positive and significant influence of temperature was found in Watamu Park. Distinct assemblages of larvae occurred within the parks indicating within park patchiness in larval distribution and, perhaps existence of retention features. These results provide useful baseline data on larval distribution in the WIO, and will be useful in understanding population dynamics of adult populations when complimented with additional oceanographic data.     

Direct proteasome binding and subsequent degradation of unspliced XBP-1 prevent its intracellular aggregation

The non-canonical splicing of XBP-1 mRNA is a hallmark of the mammalian unfolded protein response (UPR). The proteasomal degradation of unspliced XBP-1 (XBP-1u) facilitates the termination of the UPR. Thus, understanding the mechanism of XBP-1u degradation may allow control over UPR duration and intensity.We show that XBP-1u interacts with purified 20S proteasomes through its unstructured C-terminus, which leads to its degradation in a manner that autonomously opens the proteasome gate. In living cells, the C-terminus of XBP-1u accumulates in aggresome structures in the presence of proteasome inhibitors. We propose that direct proteasomal degradation of XBP-1u prevents its intracellular aggregation.

Structured summary

MINT-7302217: XBP1-u (uniprotkb:P17861-1) binds (MI:0407) to Proteasome subunit alpha 7.2 (uniprotkb:O14818) by pull down (MI:0096)MINT-7302148: Vimentin (uniprotkb:P08670) and XBP1-u (uniprotkb:P17861-1) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7302163: XBP1-u (uniprotkb:P17861-1) binds (MI:0407) to Proteasome subunit alpha 5 (uniprotkb:P28066) by pull down (MI:0096)MINT-7302186: XBP1-u (uniprotkb:P17861-1) binds (MI:0407) to Proteasome subunit alpha 6 (uniprotkb:P60900) by pull down (MI:0096)     

Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.     

PREDICT modeling and in-silico screening for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets.