Immunohistochemical Analysis of Nucleotide Excision Repair Factors XPA and XPB in Adult Rat Brain

To study the regional and cellular distribution of xeroderma pigmentosum group A and B (XPA and XPB) proteins, two nucleotide excision repair (NER) factors, in the mammalian brain we used immunohistochemistry and triple fluorescent immunostaining combined with confocal microscope scanning in brain slices of adult rat brain, including the cerebral cortex, striatum, substantia nigra compacta, ventral tegmental area, red nucleus, hippocampus, and cerebellum. Both XPA and XPB proteins were mainly expressed in neurons, because the XPA‐ or XPB‐immunopositive cells were only costained with NeuN, a specific neuronal marker, but not with glial fibrillary acidic acid, a specific astrocyte marker, in the striatum. Furthermore, XPA‐ and XPB‐positive staining were observed in the neuronal nuclei. Such subcellular distribution was consistent with the location of the NER in the cells. This study provides the first evidence that NER factors XPA and XPB exist in the nuclei of neurons in the brain, suggesting that the NER may play important roles in the process of DNA repair in adult brain neurons. Anat Rec, 291:775–780, 2008. © 2008 Wiley‐Liss, Inc.     

F-box protein FBXL19-mediated ubiquitination and degradation of the receptor for IL-33 limits pulmonary inflammation

The ST2L receptor for interleukin 33 (IL-33) mediates pulmonary inflammation and immune system-related disorders, such as asthma and rheumatoid arthritis. At present, very little is known about the molecular regulation of ST2L expression. Here we found that FBXL19, an 'orphan' member of the Skp1-Cullin-F-box family of E3 ubiquitin ligases, selectively bound to ST2L to mediate its polyubiquitination and elimination in the proteasome. Degradation of ST2L involved phosphorylation of ST2L at Ser442 catalyzed by the kinase GSK3β. Overexpression of FBXL19 abrogated the proapoptotic and inflammatory effects of IL-33 and lessened the severity of lung injury in mouse models of pneumonia. Our results suggest that modulation of the IL-33-ST2L axis by ubiquitin ligases might serve as a unique strategy for lessening pulmonary inflammation.     

Protein quality control systems associated with no‐go and nonstop mRNA surveillance in yeast

Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail‐independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl‐tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide‐dependent translation arrest as well as Ltn1‐dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail‐independent manner. Furthermore, carboxyl terminus‐truncated degradation intermediates of arrest products were stabilized in a cdc48‐3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl‐tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.     

FBXL12 regulates T‐cell differentiation in a cell‐autonomous manner

Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells including embryonic, adult tissue and cancer stem cells. The SCF^FBXL12 complex is an authentic ubiquitin ligase that targets ALDH3 for degradation. FBXL12 is essential for the differentiation of trophoblast stem cells into specific cell types in the placenta during mouse embryogenesis, but its physiological functions in adult tissues have remained unknown. We have now investigated the role of the FBXL12‐ALDH3 axis in the thymus, in which FBXL12 was most abundant among adult mouse tissues examined. During T‐cell differentiation, FBXL12 is most abundant in CD4⁺CD8⁺ (DP) cells, with its expression declining as these cells differentiate into CD4⁺CD8^? or CD4^?CD8⁺ (SP) cells. T cells of FBXL12‐null mice manifested a differentiation block at the DP–SP transition that was associated with ALDH3 accumulation in DP cells. This differentiation block was also apparent in wild‐type mouse recipients of FBXL12‐null bone marrow transplants as well as in FBXL12‐null fetal thymic organ culture, suggesting that it is a cell‐autonomous phenomenon in the thymus rather than an indirect effect of altered systemic conditions. Our results thus indicate that, in addition to its role in placental development, the FBXL12‐ALDH3 axis is required for maturation of undifferentiated thymocytes.     

Mutations at multiple CDK phosphorylation consensus sites on Cdt2 increase the affinity of CRL4Cdt2 for PCNA and its ubiquitination activity in S phase

CRL4^Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4^Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C‐terminal half region of Cdt2 (Cdt2‐18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2‐18A was colocalized with PCNA foci during S phase in comparison with Cdt2‐WT. Poly‐ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2‐18A. Other CRL4^Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2‐18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK‐mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.     

Serogroup-related escape of Yersinia enterocolitica YopE from degradation by the ubiquitin-proteasome pathway

Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.     

Successive recruitment of p‐CDC25B‐Ser351 and p‐cyclin B1‐Ser123 to centrosomes contributes to the release of mouse oocytes from prophase I arrest

Background: The molecular mechanism that controls the activation of Cyclin B1‐CDK1 complex has been widely investigated. It is generally believed that CDC25B acts as a “starter phosphatase” of mitosis. In this study, we investigate the sequential regulation of meiotic resumption by CDC25B and Cyclin B1 in mouse oocytes. Results: Injection of mRNAs coding for CDC25B‐Ser351A and/or Cyclin B1‐Ser123A shows a more potent maturation‐inhibiting ability than their respective wild type. Co‐injection of mRNAs coding for phosphor‐mimic CDC25B‐Ser351D and Cyclin B1‐Ser123D can rescue this prophase I arrest induced by CDC25B‐Ser351A or Cyclin B1‐Ser123A. In addition, p‐CDC25B‐Ser351 is co‐localized at the microtubule‐organizing centers (MTOCs) with Aurora kinase A (AURKA) during maturation and p‐Cyclin B1‐Ser123 is only captured on MTOCs shortly before germinal vesicle breakdown (GVBD). Depletion of AURKA not only resulted in metaphase I (MI) spindle defects and anaphase I (AI) abnormal chromosomes separation but also prevented the phosphorylation of CDC25B‐Ser351 at centrosomes. AURKA depletion induced deficiencies of spindle assembly and progression to MII can be rescued by CDC25B‐Ser351D mRNA injection. Conclusions: AURKA induced phosphorylation and recruitment of CDC25B to MTOCs prior to p‐Cyclin B1‐Ser123, and this sequential regulation is essential for the commitment of the oocytes to resume meiosis. Developmental Dynamics 244:110–121, 2015. © 2014 Wiley Periodicals, Inc.