Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome

BACKGROUND Angelman syndrome(AS)is caused by maternal chromosomal deletions,imprinting defects,paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations.However the genetic basis remains unclear for several patients.AIM To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing.METHODS We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021,with a strong suspicion of AS and absence of chromosomal aberrations.The UBE3A gene was screened for mutation detection.Two unrelated patients issued from consanguineous families were subjected to exome analysis.RESULTS We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C(intron14),c.2507+43T>A(Exon15)and insertion in Exon7:c.30-47_30-46.The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further.CONCLUSION Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis.Our exome findings could rise to new potential alternative target genes for genetic counseling.

The Role of E3 Ligases in Macrophage-mediated Inflammation

Macrophages,existed in almost all organs of the body,are responsible for detecting tissue injury,pathogens,playing a key role in host defense against a variety of invading pathogens triggering inflammatory responses.Emerging evidence suggests that macrophage-mediated immune responses are efficiently regulated by the ubiquitination modification,which is responsible for normal immune responses.However,numerous studies indicates that the aberrant activation or inhibition of macrophage-mediated immune responses occurs in inflammation,mainly caused by dysregulated ubiquitination modification due to E3 ubiquitin ligases mutations or abnormal expression.Notably,E3 ubiquitin ligases,responsible for recognizing the substrates,are key enzymes in the ubiquitin proteasome system(UPS)composed of ubiquitin(Ub),ubiquitin-activating E1 enzymes,ubiquitin-conjugating E2 enzymes,E3 ubiquitin ligases,26S proteasome,and deubiquitinating enzymes.Intriguingly,several E3 ubiquitin ligases are involved in the regulation of some common signal pathways in macrophage-mediated inflammation,including Toll-like receptors(TLRs),nucleotide-binding oligomerization domain(NOD)-like receptors(NLRs),RIG-I-like receptors(RLRs),C-type lectin receptors(CLRs)and the receptor for advanced glycation end products(RAGE).Herein,we summarized the physiological and pathological roles of E3 ligases in macrophage-mediated inflammation,as well as the inhibitors and agonists targeting E3 ligases in macrophage mediated inflammation,providing the new ideas for targeted therapies in macrophage-mediated inflammation caused aberrant function of E3 ligases.

Research Progress in Function and Regulation of E3 Ubiquitin Ligase SMURF1

Smad ubiquitylation regulatory factor 1(Smurf1)is an important homologous member of E6-AP C-terminus type E3 ubiquitin ligase.Initially,Smurf1 was reportedly involved in the negative regulation of the bone morphogenesis protein(BMP)pathway.After further research,several studies have confirmed that Smurf1 is widely involved in various biological processes,such as bone homeostasis regulation,cell migration,apoptosis,and planar cell polarity.At the same time,recent studies have provided a deeper understanding of the regulatory mechanisms of Smurf1’s expression,activity,and substrate selectivity.In our review,a brief summary of recent important biological functions and regulatory mechanisms of E3 ubiquitin ligase Smurf1 is proposed.

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.:MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.).Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp,encoding 424 amino acids.Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri.The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain.qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species,and was highly expressed in the root,while the expression of MdPUB29 was significantly inhibited by exogenous NaCl.Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions.Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29.The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions,suggesting that MdPUB29 may positively regulate salt tolerance.

Role of E3 ubiquitin ligases in lung cancer

E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.Therefore,E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation,proliferation and apoptosis.E3 ubiquitin ligases are often found overexpressed in human cancers,including lung cancer,and their deregulation has been shown to contribute to cancer development.However,the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting.In this review,we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer.Furthermore,we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets.By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis,we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.

Role of E3 ubiquitin ligases in gastric cancer

E3 ubiquitin ligases have an important role in carcinogenesis and include a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.So far,E3 ubiquitin ligases have been reported to have a role in a variety of biological processes including cell cycle regulation,cell proliferation,and apoptosis.Recently,several kinds of E3 ubiquitin ligases were demonstrated to be generally highly expressed in gastric cancer(GC) tissues and to contribute to carcinogenesis.In this review,we summarize thecurrent knowledge and information about the clinical significance of E3 ubiquitin ligases in GC.Bortezomib,a proteasome inhibitor,encouraged the evaluation of other components of the ubiquitin proteasome system for pharmaceutical intervention.The clinical value of novel treatment strategies targeting aberrant E3 ubiquitin ligases for GC are discussed in the review.

Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the lAP family members and is required for GIDE＇s E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE- induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over-or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.

Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.

TaGW2L,a GW2-like RING finger E3 ligase,positively regulates heading date in common wheat(Triticum aestivum L.)

RING finger E3 ligases play an important role in regulating plant growth and development by mediating substrate degradation.In this study,we identified TaGW2L,encoding a Grain width and weight2(GW2)-like RING finger E3 ligase,as a novel positive regulator of heading date in wheat(Triticum aestivum L.).TaGW2L exhibited high amino acid sequence similarities with TaGW2 homoeologs,particularly in the conserved RING finger domain.Expression analysis indicated that TaGW2L was constitutively expressed in various wheat tissues.TaGW2L showed transactivation activity in yeast and could interact with the ubiquitin-conjugating enzymes E2_(s).An in vitro ubiquitination assay verified that TaGW2L possessed a similar E3 ligase activity to TaGW2.Overexpression of the TaGW2L-7A homoeolog in wheat led to a significantly earlier heading date under both natural conditions and long-day conditions.Transcriptome analysis revealed that multiple known genes positively regulating wheat heading were significantly upregulated in the TaGW2L-7A-overexpression plants compared with the wild-type control.Together,our findings shed light on the role of TaGW2L in wheat heading date and provide potential applications of TaGW2L for the adaptation improvement of crops.

Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development,and phytohormone and abiotic stress responses

Background： RING H2 finger E3 ligase （RH2FE3） genes encode cysteine rich proteins that mediate E3 ubiquitin ligase activity and degrade target substrates. The roles of these genes in plant responses to phytohormones and abiotic stresses are well documented in various species, but their roles in cotton fiber development are poorly understood. To date, genome wide identification and expression analyses of Gossypium hirsutum RH2FE3 genes have not been reported. Methods： We performed computational identification, structural and phylogenetic analyses, chromosomal distribution analysis and estimated KJKs values of G hirsutum RH2FE3 genes. Orthologous and paralogous gene pairs were identified by all versus all BLASTP searches. We predicted cis regulatory elements and analyzed microarray data sets to generate heatmaps at different development stages. Tissue specific expression in cotton fiber, and hormonal and abiotic stress responses were determined by quantitative real time polymerase chain reaction （qRT PCR） analysis. Results： We investigated 140 G hirsutum, 80 G. orboreum, and evolutionary mechanisms and compared them with orthologs 89 G. roimondii putative RH2FB genes and their in Arobidopsis and rice. A domain based analysis of the G hirsutum RH2FE3 genes predicted conserved signature motifs and gene structures. Chromosomal localization showed the genes were distributed across all G hirsutum chromosomes, and 60 duplication events （4 tandem and 56 segmental duplications） and 98 orthologs were detected, cis elements were detected in the promoter regions of G hirsutum RH2FE3 genes. Microarray data and qRT PCR analyses showed that G hirsutum RH2FE3 genes were strongly correlated with cotton fiber development. Additionally, almost all the （brassinolide, gibberellic acid （GA）, indole 3-acetic acid drought, and salt）. dentified genes were up regulated in response to phytohormones （IAA）, and salicylic acid （SA）） and abiotic stresses （cold, heat, Conclusions： The genome wide identification, comprehensive analysis, and characterization of conserved domains and gene structures, as well as phylogenetic analysis, cis element prediction, and expression profile analysis of G hirsutum RH2FE3 genes and their roles in cotton fiber development and responses to plant hormones and abiotic stresses are reported here for the first time. Our findings will contribute to the genome wide analysis of putative RH2FE3 genes in other species and lay a foundation for future physiological and functional research on G hirsutum RH2FE3 genes.

Effects of Nephrolithiasis on Serum DNase(Deoxyribonuclease Ⅰ and Ⅱ) Activity and E3 SUMO-Protein Ligase NSE2(NSMCE2) in Malaysian Individuals

Objective Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase Ⅰ/Ⅱ activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. Methods Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase Ⅰ/Ⅱ activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. Results The result indicated that mean levels of sera NSMCE2 have a significantly increase（P〈0.01） in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase Ⅰ and Ⅱ were significantly elevated in nephrolithiasis patients（P〈0.01）. Conclusion This study suggests that an increase in serum concentrations of DNase Ⅰ/Ⅱ and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

Study the effect of kidney stones on serum xanthine oxidase, ecto-5'-nucleotidase activity and E3 SUMO-protein ligase NSE2(NSMCE2) in Malaysian individuals

Objective: To verify possible relations between 5'-nucleotidase, xanthine oxidase to E3 small ubiquitin-like modifier-protein ligase non structural maintenance of chromosomes elements 2 in sera patients with kidney stones and to evaluate the possibility of a new biomarker for the evaluation of kidney damage. Methods: A sixty patients with known kidney stones who appeared the government health clinics in Kuantan–Pahang and fifty apparently healthy were taken as control group. The 5'-nucleotidase,xanthine oxidase and other biochemical parameters were measured by colorimetric tests. The serum NSMCE2 were measured by enzyme linked immunosorbent assay.Results: The mean serum xanthine oxidase [(39.98±19.70) IU/L] and ecto-5'-nucleotidase activity(40.03±9.53 IU/L) were significantly higher than the controls' levels of(18.04 ±6.26) and(16.06 ±4.61) IU/L respectively. There were 85.00% and 83.33%, of patients with kidney stones who had abnormal ecto-5'-nucleotidase activity and uric acid respectively while xanthine oxidase activity was less sensitive 58.33%.Conclusions: The present study suggests that the increase in serum of xanthine oxidase,ecto-5'-nucleotidase activities E3 small ubiquitin-like modifier-protein ligase NSE2 concentration can be used as biomarkers for diagnosis of kidney damage in patients with kidney stone,also in developments of change DNA damage and inflammation disorders in these patients.

An atypical ubiquitin ligase at the heart of neural development and programmed axon degeneration

The degeneration of nerve fibres following injury was first described by Augustus Waller over 170 years ago.Initially assumed to be a passive process,it is now evident that axons respond to insult via regulated cellular signaling events resulting in their programmed degeneration.Pro-survival and prodegenerative factors have been identified and their regulato ry mechanisms are beginning to emerge.The ubiquitin system has been implicated in the pro-degenerative process and a key component is the ubiquitin E3 ligase MYCBP2(also known as PHR1).Ubiquitin E3 ligases are tasked with the transfer of the small protein modifier ubiquitin to substrates and consist of hundreds of members.They can be classified as single subunit systems or as multi-subunit complexes.Their catalytic domains can also be assigned to three general architectures.Hints that MYCBP2 might not conform to these established formats came to light and it is now clear from biochemical and structural studies that MYCBP2 is indeed an outlier in terms of its modus operandi.Furthermore,the unconventional way in which MYCBP2 transfe rs ubiquitin to substrates has been linked to neurodevelopmental and pro-degenerative function.Herein,we will summarize these research developments relating to the unusual features of MYCBP2 and postulate therapeutic strategies that prevent Walle rian degeneration.These have exciting potential for providing relief from pathological neuropathies and neurodegenerative diseases.

Serum E3 SUMO-protein ligase NSE2 level and peroxynitrite related to oxidative stress in nephrolithiasis patients

Objective: To prove probable relations between serum E3 SUMO-protein ligase NSE2(NSMCE2) concentration, peroxynitrite related to oxidative stress in nephrolithiasis patients.Methods: A total of 60 patients with nephrolithiasis and 50 healthy volunteers were involved in this study. Colorimetric method was used to detect blood urea, creatinine, uric acid, protein, albumin, total antioxidant status, total oxidant status, peroxynitrite, nitric oxide and oxidative stress index. Glutathione, NSMCE2 and superoxide dismutase were measured by ELISA.Results: A significant increase in level of peroxynitrite, total oxidant status, NSMCE2 and oxidative stress index in patients was observed, while total antioxidant status and glutathione were significantly decreased.Conclusions: The study concluded that serum NSMCE2 significantly correlated with peroxynitrite and oxidative stress in patients with nephrolithiasis.

Jianpi Decoction Combined with Medroxyprogesterone Acetate Alleviates Cancer Cachexia and Prevents Muscle Atrophy by Directly Inhibiting E3 Ubiquitin Ligase

ObjectiveTo provide comprehensive evidence for the anti-cancer cachexia effect of Jianpi Decoction(JP)and to explore its mechanism of anti-cancer cachexia.MethodsA mouse model of colon cancer(CT26)-induced cancer cachexia(CC)was used to investigate the anti-CC effect of JP combined with medroxyprogesterone acetate(MPA).Thirty-six mice were equally divided into 6 groups:normal control,CC,MPA(100 mg·kg^(−1)·d^(−1)),MPA+low-dose(20 mg·kg^(−1)·d^(−1))JP(L-JP),MPA+medium-dose(30 mg·kg^(−1)·d^(−1))JP(M-JP),and MPA+high-dose(40 mg·kg^(−1)·d^(−1))JP(H-JP)groups.After successful modeling,the mice were administered by gavage for 11 d.The body weight and tumor volume were measured and recorded every 2 d starting on the 8th day after implantation.The liver,heart,spleen,lung,kidney,tumor and gastrocnemius muscle of mice were collected and weighed.The pathological changes of the tumor was observed,and the cross-sectional area of the gastrocnemius muscle was calculated.The protein expressions of STAT3 and E3 ubiquitinase in the gastrocnemius muscle were measured by Western blot.In addition,an in vitro C2C12 myotube formation model was established to investigate the role of JP in hindering dexamethasone-induced muscle atrophy.In vitro experiments were divided into control,model,and JP serum groups.After 2-d administration,microscopic photographs were taken and myotube diameters were calculated.Western blot was performed to measure the protein expressions of STAT3 and E3 ubiquitinase.ResultsJP combined with MPA restored tumor-induced weight loss(P<0.05,vs.CC)and muscle fiber size(P<0.01,vs.CC).Mechanistically,JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx in tumor-induced muscle atrophy in vivo(P<0.05,vs.CC).In addition,JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx and p-STAT3 phosphorylation(P<0.05 or P<0.01 vs.model group)in C2C12 myotubes treated with dexamethasone in vitro.ConclusionsAdministration of JP combined with MPA restores tumor-induced cachexia conditions.In addition,the profound effect of JP combined with MPA on tumor-induced cachexia may be due to its inhibition of muscle proteolysis(E3 ubiquitinase system).

血清sTREM-1、sTim3、cullin4A与肺结核患者肺部受累情况的相关性

目的研究血清可溶性髓系细胞触发受体-1(sTREM-1)、可溶性T细胞免疫球蛋白黏蛋白3(sTim-3)、泛素连接酶4A(cullin4A)在反映4MRZE化疗方案的疗效及肺结核患者肺部受累情况中的价值。方法将2018年1月至2021年1月武汉市金银潭医院及赤壁市人民医院收治的265例肺结核患者根据治疗方案的不同分为采用2H3R3Z3E3/4H3R3标准四联疗法方案治疗的对照组(n=97)、4MRZE超短程化疗方案治疗的4MRZE组(n=88)和2HRZE/4HR方案治疗的2HRZE/4HR组(n=80)。比较3组治疗后临床疗效,以及治疗前和治疗2、4个月血清sTREM-1、sTim3、cullin4A水平变化,分析血清sTREM-1、sTim3、cullin4A在反映肺结核患者肺部受累情况中的价值。结果4MRZE组、2HRZE/4HR组治疗2、4个月时的痰菌转阴率及病灶有效吸收率均高于对照组(P<0.05),且4MRZE组高于2HRZE/4HR组(P<0.05);在治疗过程中,3组患者血清sTREM-1、sTim3呈下降趋势,而cullin4A mRNA呈上升趋势,且4MRZE组患者治疗2、4个月的血清sTREM-1、sTim3显著低于2HRZE/4HR组、对照组(P<0.05),而cullin4A mRNA显著高于2HRZE/4HR组、对照组(P<0.05)。但是治疗完成后,3组痰菌转阴率、病灶有效吸收率,以及血清sTREM-1、sTim3水平比较,差异无统计学意义(P>0.05)。结论4MRZE化疗方案在肺结核治疗中具备治疗周期短的同时保证治疗效果良好的优点。血清sTREM-1、sTim3、cullin4A水平在反映痰菌转阴及病灶有效吸收中具有一定价值。

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands;however,such mechanisms regulating nodulation factor(NF)receptor(NFR)-mediated perception of NFs to establish symbiosis remain unclear.In this study,we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1(NIRE1)in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus.We demonstrated that NiRE1 has a dual function in this regulatory process.It associates with both NFR1 and NFR5,facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation.However,following rhizobial inoculation,NFR1 phosphorylates NIRE1ata conserved residue,Tyr-109,inducing a functional switch in NIRE1,which enables NIRE1tomediateK63-linkedpolyubiquitination,thereby stabilizing NFR1/NFR5 in infected root cells.The introduction of phospho-dead NIRE1Y1osF leads to delayed nodule development,underscoring the significance of phosphorylation at Tyr-1o9 in orchestrating symbiotic processes.Conversely,expression of the phospho-mimic NIRE1Y0E results in the formation of spontaneous nodules in L.japonicus,further emphasizing the critical role of the phosphorylation-dependent functional switch in NiRE1.In summary,these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylationdependent functional switch to dynamically and precisely regulate NF receptor protein levels.

射血分数保留心力衰竭患者血清WWP1和NLRP3的表达水平及其临床价值研究

目的 探讨WW结构域E3泛素蛋白连接酶1(WW domain-containing E3 ubiquitin protein ligase 1,WWP1)和核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)在射血分数保留心力衰竭(heart failure with preserved ejection fraction,HFpEF)患者血清中的表达水平及临床意义。方法选取华北医疗健康集团峰峰总医院2021年1月~2022年9月收治的153例HFpEF患者为观察组,并根据患者纽约心脏病协会(New York Heart Association,NYHA)心功能分级分为心功能分级Ⅰ~Ⅱ级组(n=64)和心功能分级Ⅲ~Ⅳ级组(n=89),另选取同期体检健康的148例志愿者为对照组。血清WWP1,NLRP3水平与患者心功能指标的相关性采用Pearson分析;受试者工作特征(receiver operating characteristic,ROC)曲线分析血清WWP1和NLRP3水平对HFpEF患者心衰严重程度的诊断价值。结果 与对照组比较,观察组血清WWP1(1.68±0.35 vs 1.04±0.19)和NLRP3(6.72±1.26 ng/ml vs 4.57±0.84 ng/ml)表达水平明显升高,差异具有统计学意义(t=19.623,17.359,均P <0.05);与心功能分级Ⅰ~Ⅱ级组比较,心功能分级Ⅲ~Ⅳ级组血清WWP1(1.87±0.39 vs 1.42±0.32)和NLRP3(7.53±1.40 ng/ml vs 5.59±1.18 ng/ml)表达水平明显升高,差异具有统计学意义(t=7.744,9.017,均P<0.05);心功能分级Ⅰ~Ⅱ级组与心功能分级Ⅲ~Ⅳ级组心率、左心房内径(left atrial diameter,LAD)、左心室舒张末期内径(left ventricular end-diastolic diameter,LVEDD)、左室舒张末期后壁厚度(left ventricular end-diastolic posterior wall thickness,LVPWT)、左心室射血分数(left ventricular ejection fraction,LVEF)、二尖瓣舒张早期流速峰值(peak mitral early diastolic velocity,E)/舒张晚期流速峰值(peak late diastolic velocity,A)以及心房颤动发生率比较差异均具有统计学意义(t/χ^(2)=2.757~7.069,均P <0.05);HFpEF患者血清WWP1水平与LAD,LVEDD,LVPWT呈正相关(r=0.547,0.471,0.536,均P <0.05),与LVEF和E/A呈负相关(r=-0.485,-0.417,均P <0.05);血清NLRP3水平与LAD,LVEDD,LVPWT呈正相关(r=0.534,0.494,0.520,均P <0.05),与LVEF和E/A呈负相关(r=-0.462,-0.523,均P <0.05)。ROC结果显示,血清WWP1和NLRP3水平单独诊断HFpEF患者心衰严重程度的曲线下面积(area under the curve,AUC)分别为0.825和0.855,两者联合诊断的AUC(0.924)显著大于血清WWP1和NLRP3水平单独诊断的AUC(Z=3.600,P<0.001;Z=3.053,P=0.002)。结论 血清WWP1和NLRP3水平在HFpEF患者中明显升高,且与患者心功能密切相关,血清WWP1和NLRP3对HFpEF患者心衰严重程度具有一定的诊断价值。