A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses

Optimization of WAVE2 complex–induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac

WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP3-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP3

Estimation of Genetic Parameters, and Genetic and Environmental Effects on Progeny Carcass Traits of a Nucleus Breeding Population in Japanese Black Cattle

Genetic parameters of body weight at the start of fattening (BSF), carcass weight (CWT), subcutaneous fat thickness (SFT), rib thickness (RBT), meat quality grade (MQG), beef marbling score (BMS) and rib eye area (REA) in Japanese Black cattle were estimated. The effects of genetic and environmental factors on fattening performance and carcass quality traits of the progeny were also analyzed. The averages of BSF, CWT, SFT, RBT and REA were 189 kg, 408 kg, 24.9 mm, 69.3 mm and 47.5 cm^2, respectively. CWT was significantly affected (p<0.01) by sire, while BSF (p<0.01), CWT (p<0.01), SFT (p<0.01), RBT (p<0.01) and REA (p<0.05) were significantly affected by dam, indicating the scope of the applicability of selective breeding for improving the dam population for producing beef cattle with greater genetic ability to carcass traits. The sex of the calf had a significant (p<0.01 or p<0.05) effect on all the traits studied except REA. BSF, RBT, MQG and BMS were significantly (p<0.01) affected by the year and season of the birth of calves. The fattening farm exerted a significant influence (p<0.01) on BSF, CWT, MQG and BMS. The heritability estimates for BMS, CWT, RBT, BSF and MQG were 0.36, 0.34, 0.31, 0.26 and 0.23, respectively. Genetically, REA correlated negatively with BSF (-0.57) and SFT (-0.69), as did BMS with CWT (-0.35), SFT (-0.50) and RBT (-0.44). All the other traits correlated positively with one another

High defect stage, contralateral defects, and poor flexibility are negative predictive factors of bone union in pediatric and adolescent athletes with spondylolysis

Purpose : To identify predisposition to spondylolysis and physical characteristics associated with “bone union” following conservative spondylolysis treatment among pediatric and adolescent athletes. Methods : We retrospectively analyzed pediatric and adolescent athletes with spondylolysis who underwent conservative treatment and rehabilitation for three or more consecutive months following sports activity cessation. Patients with terminal spondylolysis or who did not discontinue sports activities were excluded. We compared physical fitness factors in the union and nonunion groups and examined the association between bone union and spondylolysis severity by logistic regression analysis. Results : Of 183 patients with spondylolysis who underwent rehabilitation over a four-year period, 127 patients with 227 defects were included in the final analysis. Bone union was achieved in 66.5% (151/227) of the pars interarticularis defects and 70.1% (89/127) of the patients. On multivariate analysis, stage of pars interarticularis defect (odds ratio [OR], 0.26 ; p = 0.0027), stage of contralateral pars interarticularis defect (OR, 0.51 ; p = 0.00026), and straight leg-raising test (OR, 1.06 ; p = 0.028) were significantly associated with bone union. Conclusions : High defect stage, stage of the contralateral pars interarticularis defect, and poor flexibility were negative prognostic factors of bone healing in athletes with spondylolysis

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

Kato O, Youn J-W, Stansen KC, Matsui D, Oikawa T, Wendisch VF. Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate. BMC Microbiology. 2010;10(1): 321.Background: Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Results: Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer. Conclusions: Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1−/−-mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways

Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins

Chaperone protein BiP binds to Ire1 and dissociates in response to endoplasmic reticulum (ER) stress. However, it remains unclear how the signal transducer Ire1 senses ER stress and is subsequently activated. The crystal structure of the core stress-sensing region (CSSR) of yeast Ire1 luminal domain led to the controversial suggestion that the molecule can bind to unfolded proteins. We demonstrate that, upon ER stress, Ire1 clusters and actually interacts with unfolded proteins. Ire1 mutations that affect these phenomena reveal that Ire1 is activated via two steps, both of which are ER stress regulated, albeit in different ways. In the first step, BiP dissociation from Ire1 leads to its cluster formation. In the second step, direct interaction of unfolded proteins with the CSSR orients the cytosolic effector domains of clustered Ire1 molecules

Identification of a consensus element recognized and cleaved by IRE1α

IRE1α is an endoplasmic reticulum (ER)-located transmembrane RNase that plays a central role in the ER stress response. Upon ER stress, IRE1α is activated and cleaves specific exon–intron sites in the mRNA encoding the transcription factor X-box-binding protein 1 (XBP1). In addition, previous studies allow us to predict that IRE1α targets several RNAs other than the XBP1. In fact, we have identified CD59 mRNA as a cleavage target of IRE1α. However, it is not yet clear how IRE1α recognizes and cleaves target RNAs. To address this question, we devised a unique method that combines an in vitro cleavage assay with an exon microarray analysis, and performed genome-wide screening for IRE1α cleavage targets. We identified 13 novel mRNAs as candidate IRE1α cleavage targets. Moreover, an analysis of the novel cleavage sites revealed a consensus sequence (CUGCAG) which, when accompanied by a stem-loop structure, is essential for IRE1α-mediated cleavage. The sequence and structure were also conserved in the known IRE1α cleavage targets, CD59 and XBP1. These findings provide the important clue to understanding the molecular mechanisms by which IRE1α recognizes and cleaves target RNAs