Evidence for a phosphorylation-independent role for Ser 32 and 36 in proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in B cells

Constitutive NF-kappaB activity has emerged as an important cell survival regulator. Canonical inducible NF-kappaB activation involves IkappaB kinase (IKK)-dependent dual phosphorylation of Ser 32 and 36 of IkappaBalpha to cause its beta-TrCP-dependent ubiquitylation and proteasomal degradation. We recently reported that constitutive NF-kappaB (p50/c-Rel) activity in WEHI231 B cells is maintained through proteasome inhibitor-resistant (PIR) IkappaBalpha degradation in a manner that requires Ser 32 and 36, without the requirement of a direct interaction with beta-TrCP. Here we specifically examined whether dual phosphorylation of Ser 32 and 36 was required for PIR degradation. Through mutagenesis studies, we found that dual replacement of Ser 32 and 36 with Glu permitted beta-TrCP and proteasome-dependent, but not PIR, degradation. Moreover, single replacement of either Ser residue with Leu permitted PIR degradation in WEHI231 B cells. These results indicate that PIR degradation occurs in the absence of dual phosphorylation, thereby explaining the beta-TrCP-independent nature of the PIR pathway. Additionally, we found evidence that PIR IkappaBalpha degradation controls constitutive NF-kappaB activation in certain multiple myeloma cells. These results suggest that B lineage cells can differentiate between PIR and canonical IkappaBalpha degradation through the absence or presence of dually phosphorylated IkappaBalpha.     

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1 cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1 in pancreatic β cells. Expression of collectrin was decreased in the islets of HNF-1 (−/−) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the β cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca²⁺ influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.     

Quantification of Cellular NEMO Content and Its Impact on NF-κB Activation by Genotoxic Stress

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 10⁵ molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6–6x10⁵ molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

A GC polymorphism associated with serum 25-hydroxyvitamin D level is a risk factor for hip fracture in Japanese patients with rheumatoid arthritis: 10-year follow-up of the Institute of Rheumatology,Rheumatoid Arthritis cohort study

Introduction

Vitamin D deficiency has been reported to be common in patients with rheumatoid arthritis (RA) who have a higher prevalence of osteoporosis and hip fracture than healthy individuals. Genetic variants affecting serum 25-hydroxyvitamin D (25(OH)D) concentration, an indicator of vitamin D status, were recently identified by genome-wide association studies of Caucasian populations. The purpose of this study was to validate the association and to test whether the serum 25(OH)D-linked genetic variants were associated with the occurrence of hip fracture in Japanese RA patients.

Methods

DNA samples of 1,957 Japanese RA patients were obtained from the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort DNA collection. First, five single nucleotide polymorphisms (SNPs) that were reported to be associated with serum 25(OH)D concentration by genome-wide association studies were genotyped. The SNPs that showed a significant association with serum 25(OH)D level in the cross-sectional study were used in the longitudinal analysis of hip fracture risk. The genetic risk for hip fracture was determined by a multivariate Cox proportional hazards model in 1,957 patients with a maximum follow-up of 10 years (median, 8 years).

Results

Multivariate linear regression analyses showed that rs2282679 in GC (the gene encoding group-specific component (vitamin D binding protein)) locus was significantly associated with lower serum 25(OH)D concentration (P = 8.1 × 10^-5). A Cox proportional hazards model indicated that rs2282679 in GC was significantly associated with the occurrence of hip fracture in a recessive model (hazard ratio (95% confidence interval) = 2.52 (1.05-6.05), P = 0.039).

Conclusions

A two-staged analysis demonstrated that rs2282679 in GC was associated with serum 25(OH)D concentration and could be a risk factor for hip fracture in Japanese RA patients.     

Suppression of α‐amylase genes improves quality of rice grain ripened under high temperature

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism‐related genes. Here we report the involvement of a starch‐hydrolyzing enzyme, α‐amylase, in high temperature‐triggered grain chalkiness. In developing seeds, high temperature induced the expression of α‐amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α‐amylase activity, while it decreased an α‐amylase‐repressing plant hormone, ABA, suggesting starch to be degraded by α‐amylase in developing grains under elevated temperature. Furthermore, RNAi‐mediated suppression of α‐amylase genes in ripening seeds resulted in fewer chalky grains under high‐temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α‐amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.     

Two new taxa of Achnanthidium and Encyonema (Bacillariophyceae) from the Yom River,Thailand, with special reference to the areolae occlusions implying ontogenetic relationship

We discovered two new taxa of Achnanthidium and Encyonema occurring with high abundance in the upstream of the Yom River, which is one of four major rivers in Northern Thailand. Achnanthidium pseudoconspicuum var. yomensis var. nov. closely resembles Achnanthidium pseudoconspicuum var. pseudoconspicuum in scanning electron microscopy (SEM), but has clearly different valve morphometry under the light microscopy (LM). Achnanthidium pseudoconspicuum var. yomensis is also similar to A. japonicum in valve morphology under LM, but differs to this species based on SEM observation. Encyonema yuwadeeanum sp. nov. shows similarities to Encyonema leei and Encyonema subkukenanum, but differs in valve shape and/or polar fissure shape. We discuss the possible valve ontogenetic relationship between Achnanthidium and Cymbellales based on the detailed areolae structures of these two new taxa.     

Identification of cis-localization elements of the maize 10-kDa δ-zein and their use in targeting RNAs to specific cortical endoplasmic reticulum subdomains

The RNAs for the storage proteins of rice ( Oryza sativa ), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis -localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis -localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis -elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa δ-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis -localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis -localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.     

Effects of pericardial constraint and ventricular interaction on left ventricular hemodynamics in the unloaded heart

Pericardial constraint and ventricular interaction influence left ventricular (LV) performance when preload is high. However, it is unclear if these constraining forces modulate LV filling when the heart is unloaded, such as during upright posture, in humans. Fifty healthy individuals underwent right heart catheterization to measure pulmonary capillary wedge (PCWP) and right atrial pressure (RAP). To evaluate the effects of pericardial constraint on hemodynamics, transmural filling pressure (LVTMP) was defined as PCWP-RAP. Beat-to-beat blood pressure (BP) waveforms were recorded, and stroke volume (SV) was derived from the Modelflow method. After measurements at -30 mmHg lower body negative pressure (LBNP), which approximates the upright position, LBNP was released, and beat-to-beat measurements were performed for 15 heartbeats. At -30 mmHg LBNP, RAP and PCWP were significantly decreased. During the first six beats of LBNP release, heart rate (HR) was unchanged, while BP increased from the fourth beat. RAP increased faster than PCWP resulting in an acute decrease in LVTMP from the fourth beat. A corresponding drop in SV by 3% was observed with no change in pulse pressure. From the 7th to 15th beats, LVTMP and SV increased steadily, followed by a decreased HR due to the baroreflex. A decreased TMP, but not PCWP, caused a transient drop in SV with no changes in HR or pulse pressure during LBNP release. These results suggest that the pericardium constrains LV filling during LBNP release, enough to cause a small but significant drop of SV, even at low cardiac filling pressure in healthy humans.     

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.