Polyubiquitination inhibition of estrogen receptor alpha and its implications in breast cancer

Estrogen receptor alpha(ERα) is detected in more than 70% of the cases of breast cancer. Nuclear activity of ERα, a transcriptional regulator, is linked to the development of mammary tumors, whereas the extranuclear activity of ERα is related to endocrine therapy resistance. ERα polyubiquitination is induced by the estradiol hormone, and also by selective estrogen receptor degraders, resulting in ERα degradation via the ubiquitin proteasome system. Moreover, polyubiquitination is related to the ERα transcription cycle, and some E3-ubiquitin ligases also function as coactivators for ERα. Several studies have demonstrated that ERα polyubiquitination is inhibited by multiple mechanisms that include posttranslational modifica-tions, intera-ctions with coregula-tors, a-nd forma-tion of specific protein complexes with ERα. These events are responsible for an increase in ERα protein levels and deregulation of its signaling in breast cancers. Thus, ERα polyubiquitination inhibition may be a key factor in the progression of breast cancer and resistance to endocrine therapy.

Polyubiquitination and Proteasome Signals in Tubulobulbar Complexes of Rat Late Spermatids

To illustrate the involvement of tubulobulbar complexes (TBC) in ubiquitin-proteasome degradation of unnecessary proteins in the head cytoplasm of late spermatids, the localization of polyubiquitin and proteasome was studied by immunofluorescence and immunoelectron microscopy. Polyubiquitin localized to TBC and proteasome subunit α to dense materials surrounding the TBC in the cytoplasm of Sertoli cell enwrapping sickle-shaped spermatid heads. The results suggest that the TBC is a structural device for ubiquin-proteasome degradation of unnecessary proteins in the cytoplasm of spermatid head during rapid reduction of the head cytoplasm and nuclear compaction of late spermatids.

铁皮石斛多聚泛素基因Polyubiquitin1(DoUb1)的克隆及表达分析

采用RACE(rapid amplification of cDNA ends)技术获得铁皮石斛(Dendrobium officinale Kimura et Migo)Polyubiquitin1(DoUb1),该基因全长1985 bp,开放阅读框为1371 bp,编码457个氨基酸残基的亲水性蛋白,分子质量为51 179.7 ku,理论等电点p I为7.76;亚细胞定位分析表明该基因产物主要在细胞质或线粒体基质中发挥作用。DoUb1与水稻、玉米及谷子等单子叶植物的多聚泛素基因亲缘关系较近。实时荧光定量PCR(RT-qPCR)分析结果显示,DoUb1在铁皮石斛幼苗根和叶中表达量较高,茎中表达量较低;在干旱胁迫和温度胁迫过程中的表达量都低于正常状况,在干旱胁迫的早期,表达量降低,6 h时达到最低,之后随胁迫时间的延长而缓慢升高;在温度胁迫情况下,DoUb1具有与干旱胁迫相近的响应特性,但低温胁迫相比高温胁迫下,DoUb1表达量更低,表明该基因对低温胁迫更敏感,结果为后期研究铁皮石斛生长发育提供一定基础。

Expression Analysis of Polyubiquitin Genes from Bean in Response to Heavy Metals

Using differential screening of a leaf cDNA library prepared from a bean cultivar ( Phaseolus vulgaris L. cv. Saxa) exposed to HgCl 2, the authors have isolated and characterized two heavy metal_regulated cDNA fragments, designated as PvSR 5 and PvSR 51 ( Phaseolus vulgaris stress_related gene). The sequences of the cDNA inserts and homological analysis showed that both PvSR 5 and PvSR 51 encode a polyubiquitin respectively. The polyubiquitin genes were constitutively expressed in roots but weakly expressed in stems and leaves. Northern blot analysis revealed a low level of transcripts of polyubiquitin in unstressed bean leaves, but the gene expression was strongly stimulated by heavy metals, elevated temperature and salicylic acid, whereas wounding had almost no effect. These suggested that polyubiquitin might play important roles in resistance to heavy metals and various environmental stresses.

Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight

Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci （QTLs）. We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

直接观察到了蛋白折叠的轨迹

Columbia大学的J．m．Fennandez和HongbinLi首次直接观察到一种蛋白质的折叠轨迹。他们把一个由9个小蛋白ubiquitin缠绕而成的polyubiquitin固定在原子力显微镜悬臂端和一个表面之间。通过将表面拖离悬臂端的办法使polyubiquitin链机械地发生解折叠之后，逐渐降低拉伸力，同时观察链中的每个ubiquitin的重新折叠过程。因为ubiquitin的解折叠过程所经过的步骤是精确限定的，而且是不连续的，

Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling

The NS5A non-structural protein of classical swine fever virus(CSFV)is a multifunctional protein involved in viral genomic replication,protein translation,assembly of infectious virus particles,and regulation of cellular signaling pathways.Previous report showed that NS5A inhibited nuclear factor kappa B(NF-κB)signaling induced by poly(I:C);however,the mechanism involved has not been elucidated.Here,we reported that NS5A directly interacted with NF-κB essential modulator(NEMO),a regulatory subunit of the IκB kinase(IKK)complex,to inhibit the NF-κB signaling pathway.Further investigations showed that the zinc finger domain of NEMO and the aa 126–250 segment of NS5A are essential for the interaction between NEMO and NS5A.Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO.Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation.In addition,NS5A blocked the K63-linked polyubiquitination of NEMO,thus inhibiting IKK phosphorylation,IκBαdegradation,and NF-κB activation.These findings revealed a novel mechanism by which CSFV inhibits host innate immunity,which might guide the drug design against CSFV in the future.

MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade

The adaptor molecule MAVS forms prion-like aggregates to govern the RIG-I-like receptor(RLR)signaling cascade.Lys63(K63)-linked polyubiquitination is critical for MAVS aggregation,yet the underlying mechanism and the corresponding E3 ligases and deubiquitinating enzymes(DUBs)remain elusive.Here,we found that the K63-linked polyubiquitin chains loaded on MAVS can be directly recognized by RIG-I to initiate RIG-I-mediated MAVS aggregation with the prerequisite of the CARDRIG-I-CARDMAVS interaction.Interestingly,many K63-linked polyubiquitin chains attach to MAVS via an unanchored linkage.We identified Ube2N as a major ubiquitin-conjugating enzyme for MAVS and revealed that Ube2N cooperates with the E3 ligase Riplet and TRIM31 to promote the unanchored K63-linked polyubiquitination of MAVS.In addition,we identified USP10 as a direct DUB that removes unanchored K63-linked polyubiquitin chains from MAVS.Consistently,USP10 attenuates RIG-I-mediated MAVS aggregation and the production of type I interferon.Mice with a deficiency in USP10 show more potent resistance to RNA virus infection.Our work proposes a previously unknown mechanism for the activation of the RLR signaling cascade triggered by MAVS-attached unanchored K63-linked polyubiquitin chains and establishes the DUB USP10 and the E2:E3 pair Ube2N-Riplet/TRIM31 as a specific regulatory system for the unanchored K63-linked ubiquitination and aggregation of MAVS upon viral infection.

The membrane-associated E3 ubiquitin ligase MARCH3 downregulates the IL-6 receptor and suppresses colitis-associated carcinogenesis

The IL-6-STAT3 axis is critically involved in inflammation-associated carcinogenesis(IAC).How this axis is regulated to modulate IAC remains unknown.Here,we show that the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates STAT3 activation triggered by IL-6,as well as another IL-6 subfamily member,Oncostatin M(OSM).MARCH3 is associated with the IL-6 receptorα-chain(IL-6Rα)and its coreceptor gp130.Biochemical experiments indicated that MARCH3 mediates the polyubiquitination of IL-6Rαat K401 and gp130 at K849 following IL-6 stimulation,leading to their translocation to and degradation in lysosomes.MARCH3 deficiency increases IL-6-and OSM-triggered activation of STAT3 and induction of downstream effector genes in various cell types.MARCH3 deficiency enhances dextran sulfate sodium(DSS)-induced STAT3 activation,increases the expression of inflammatory cytokines,and exacerbates colitis,as well as azoxymethane(AOM)/DSS-induced colitis-associated cancer in mice.In addition,MARCH3 is downregulated in human colorectal cancer tissues and associated with poor survival across different cancer types.Our findings suggest that MARCH3 is a pivotal negative regulator of IL-6-induced STAT3 activation,inflammation,and inflammation-associated carcinogenesis.

The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target IL-5 receptor alpha to negatively regulate eosinophilic airway inflammation

Interleukin 5(IL-5)plays crucial roles in type 2-high asthma by mediating eosinophil maturation,activation,chemotaxis and survival.Inhibition of IL-5 signaling is considered a strategy for asthma treatment.Here,we identified MARCH2 and MARCH3 as critical negative regulators of IL-5-triggered signaling.MARCH2 and MARCH3 associate with the IL-5 receptorαchain(IL-5Rα)and mediate its K27-linked polyubiquitination at K379 and K383,respectively,and its subsequent lysosomal degradation.Deficiency of MARCH2 or MARCH3 modestly increases the level of IL-5Rαand enhances IL-5-induced signaling,whereas double knockout of MARCH2/3 has a more dramatic effect.March2/3 double knockout markedly increases the proportions of eosinophils in the bone marrow and peripheral blood in mice.Double knockout of March2/3 aggravates ovalbumin(OVA)-induced eosinophilia and causes increased inflammatory cell infiltration,peribronchial mucus secretion and production of Th2 cytokines.Neutralization of Il-5 attenuates OVA-induced airway inflammation and the enhanced effects of March2/3 double deficiency.These findings suggest that MARCH2 and MARCH3 play redundant roles in targeting IL-5Rαfor degradation and negatively regulating allergic airway inflammation.

Haploinsufficiency oftheTDP43 ubiquitin E3 ligase RNF220 leads to ALS-like motor neuron defects in the mouse

TDP43 pathology is seen in a large majority of amyotrophic lateral sclerosis(ALS)cases,suggesting a central pathogenic role of this regulatory protein.Clarifying the molecular mechanism controlling TDP43 stability and subcellular location might provide important insights into ALS therapy.The ubiquitin E3 ligase RNF220 is involved in different neural developmental processes through various molecular targets in the mouse.Here,we report that the RNF2207 mice showed progressively decreasing mobility to different extents,some of which developed typical ALS pathological characteristics in spinal motor neurons,including TDP43 cytoplasmic accumulation,atrocytosis,muscle denervation,and atrophy.Mechanistically,RNF220 interacts with TDP43 in vitro and in vivo and promotes its polyubiquitination and proteasomal degradation.In conclusion,we propose that RNF220 might be a modifier of TDP43 function in vivo and contribute to TDP43 pathology in neurodegenerative disease like ALS.