Ubiquitin Pathway Research Articles

Selective binding of retrotransposons by ZFP352 facilitates the timely dissolution of totipotency network

Acquisition of new stem cell fates relies on the dissolution of the prior regulatory network sustaining the existing cell fates. Currently, extensive insights have been revealed for the totipotency regulatory network around the zygotic genome activation (ZGA) period. However, how the dissolution of the totipotency network is triggered to ensure the timely embryonic development following ZGA is largely unknown. In this study, we identify the unexpected role of a highly expressed 2-cell (2C) embryo specific transcription factor, ZFP352, in facilitating the dissolution of the totipotency network. We find that ZFP352 has selective binding towards two different retrotransposon sub-families. ZFP352 coordinates with DUX to bind the 2C specific MT2_Mm sub-family. On the other hand, without DUX, ZFP352 switches affinity to bind extensively onto SINE_B1/Alu sub-family. This leads to the activation of later developmental programs like ubiquitination pathways, to facilitate the dissolution of the 2C state. Correspondingly, depleting ZFP352 in mouse embryos delays the 2C to morula transition process. Thus, through a shift of binding from MT2_Mm to SINE_B1/Alu, ZFP352 can trigger spontaneous dissolution of the totipotency network. Our study highlights the importance of different retrotransposons sub-families in facilitating the timely and programmed cell fates transition during early embryogenesis.

UBE2C expression is elevated in hepatoblastoma and correlates with inferior patient survival.

Hepatoblastoma (HB) is the most common malignant liver tumor among children. To gain insight into the pathobiology of HB, we performed RNA sequence analysis on 5 patient-derived xenograft lines (HB-243, HB-279, HB-282, HB-284, HB-295) and 1 immortalized cell line (HUH6). Using cultured hepatocytes as a control, we found 2,868 genes that were differentially expressed in all of the HB lines on mRNA level. The most upregulated genes were ODAM, TRIM71, and IGDCC3, and the most downregulated were SAA1, SAA2, and NNMT. Protein-protein interaction analysis identified ubiquitination as a key pathway dysregulated in HB. UBE2C, encoding an E2 ubiquitin ligase often overexpressed in cancer cells, was markedly upregulated in 5 of the 6 HB cell lines. Validation studies confirmed UBE2C immunostaining in 20 of 25 HB tumor specimens versus 1 of 6 normal liver samples. The silencing of UBE2C in two HB cell models resulted in decreased cell viability. RNA sequencing analysis showed alterations in cell cycle regulation after UBE2C knockdown. UBE2C expression in HB correlated with inferior patient survival. We conclude that UBE2C may hold prognostic utility in HB and that the ubiquitin pathway is a potential therapeutic target in this tumor.

Germ-cell-specific transcriptome analysis illuminates the chromatin and ubiquitin pathway in autism spectrum disorders.

Accumulating epidemiological studies have suggested a positive association between advanced paternal age at conception and the increased risk of neurodevelopmental outcomes such as autism spectrum disorder (ASD) in their children. Recent biological studies using human sperm have identified increased de novo mutations in aged fathers, and hyper- or hypomethylation has been identified in the sperm from aged rodents. Dysregulation of DNA methylation in sperm may explain the transgenerational effects on the pathogenesis of ASD. However, compared to these epigenetic changes in the sperm of aged males, the effects of inherited predisposition from germ cells are largely unknown. Here, we use single-cell transcriptome data sets from 13 cell lines, including 12 ASD-associated CNVs models and control, that are performed neural differentiation from mouse embryonic stem cells. This study performed comprehensive bioinformatic analyses such as gene ontology (GO), network, pathway, and upstream regulator analyses. Through these analyses, we identify several susceptible pathways, such as chromatin and ubiquitin, in addition to translational and oxidative phosphorylation. Our results suggest that dysregulation of epigenetic chromosome remodeling and ubiquitin-proteasome pathway in the germ cell is a possible modulator for subsequent differentiated cells, sperm, and egg, as a risk factor for the neurodevelopmental disorder.

The F-box protein SHORT PRIMARY ROOT modulates primary root meristem activity by targeting SEUSS-LIKE protein for degradation in rice.

Root meristem activity is essential for root morphogenesis and adaptation, but the molecular mechanism regulating root meristem activity is not fully understood. Here, we identify an F-box family E3 ubiquitin ligase named SHORT PRIMARY ROOT (SHPR) that regulates primary root (PR) meristem activity and cell proliferation in rice. SHPR loss-of-function mutations impair PR elongation in rice. SHPR is involved in the formation of an SCF complex with the Oryza sativa SKP1-like protein OSK1/20. We show that SHPR interacts with Oryza sativa SEUSS-LIKE (OsSLK) in the nucleus and is required for OsSLK polyubiquitination and degradation by the ubiquitin 26S-proteasome system (UPS). Transgenic plants overexpressing OsSLK display a shorter PR phenotype, which is similar to the SHPR loss-of-function mutants. Genetic analysis suggests that SHPR promotes PR elongation in an OsSLK-dependent manner. Collectively, our study establishes SHPR as an E3 ubiquitin ligase that targets OsSLK for degradation, and uncovers a protein ubiquitination pathway as a mechanism for modulating root meristem activity in rice.

Human OTULIN mutations can cause distinct inflammatory disease phenotypes depending on the protein domain that is mutated

Abstract Linear ubiquitination is a post-translational modification that controls many immune signaling pathways. Linear ubiquitin chains are added to targets by LUBAC and removed by OTULIN, thus the opposing actions of LUBAC and OTULIN are critical for immune homeostasis. To understand mechanisms of how ubiquitination controls inflammation, we leveraged the use of biospecimens from patients with a novel bi-allelic OTULIN mutation. These patients have pyoderma gangrenosum (PG), an extremely rare inflammatory skin disease. OTULIN mutations have been previously described to cause a systemic autoinflammatory disease called ORAS, yet these patients define a new monogenic disease. We sought to understand this discrepancy through functional analyses of OTULIN mutations that culminate in PG vs ORAS. ORAS-causing OTULIN mutations impair the catalytic domain. However, the mutation in PG patients affects the domain responsible for binding LUBAC. Further, OTULIN’s ability to downregulate linear ubiquitin or NF-kB activity was unaffected. Patients’ transcriptional signatures showed elevated expression of genes involved in neutrophil activation and antigen presentation, and reduced expression of genes involved in Th17 differentiation, TLR signaling, and NOD-like receptor signaling. Deep immunophenotyping by CyTOF revealed that PG patients have higher frequencies of T cells and subtle defects of B cells and myeloid cells. These patients’ unique phenotype suggests an unrecognized function for OTULIN in skin inflammation. This discovery adds to the emerging spectrum of human immune-mediated diseases caused by defects in the ubiquitin pathway.

Translation inhibitor PDCD4 promotes CD8+ T cell proliferation during antiviral response

Abstract CD8+ T cell clonal expansion is critical for controlling intracellular infections and tumors. Thus, unravelling the mechanisms that regulate CD8+ T cell proliferation are imperative to improve vaccine design and immunotherapy. However, the molecular mechanisms that govern CD8+ T cell proliferation are still not fully understood. PDCD4 (Programmed cell death 4) was shown to inhibit translation by sequestering mRNA helicase eIF4A, which leads to translation inhibition of genes involved in cell proliferation. Despite the well-characterized anti-proliferative role of PDCD4, our studies suggest that PDCD4 promotes CD8+ T cell proliferation during the expansion phase of acute LCMV infection. We find that PDCD4 over-expressed Ag-specific CD8+ T cells have higher proliferation rate which leads to increased cell number compared to control Ag-specific CD8+ T cells. Consistent with the over-expression results, PDCD4 knocking-down Ag-specific CD8+ T cells undergo less cell proliferation, resulting in reduced clonal expansion compared to controls. Interestingly, contrary to the paradigm that increased proliferation generates more short-lived effector cells, CD8+ T cell proliferation mediated by PDCD4 does not skew the differentiation of short-lived and memory precursor effector cells. This PDCD4 mediated CD8+ T cell proliferation is also confirmed by over-expression of a constitutively active form of PDCD4 which resists degradation by CD8+ T cell endogenous ubiquitin pathway. In conclusion, our work has identified a novel pro-proliferative role of PDCD4 during CD8+ T cell response. Supported by grants from NIH (R01 AI139675)

The Involvement of Ubiquitination and SUMOylation in Retroviruses Infection and Latency.

Retroviruses, especially the pathogenic human immunodeficiency virus type 1 (HIV-1), have severely threatened human health for decades. Retroviruses can form stable latent reservoirs via retroviral DNA integration into the host genome, and then be temporarily transcriptional silencing in infected cells, which makes retroviral infection incurable. Although many cellular restriction factors interfere with various steps of the life cycle of retroviruses and the formation of viral latency, viruses can utilize viral proteins or hijack cellular factors to evade intracellular immunity. Many post-translational modifications play key roles in the cross-talking between the cellular and viral proteins, which has greatly determined the fate of retroviral infection. Here, we reviewed recent advances in the regulation of ubiquitination and SUMOylation in the infection and latency of retroviruses, focusing on both host defense- and virus counterattack-related ubiquitination and SUMOylation system. We also summarized the development of ubiquitination- and SUMOylation-targeted anti-retroviral drugs and discussed their therapeutic potential. Manipulating ubiquitination or SUMOylation pathways by targeted drugs could be a promising strategy to achieve a "sterilizing cure" or "functional cure" of retroviral infection.

Inhibition of USP14 promotes TNFα-induced cell death in head and neck squamous cell carcinoma (HNSCC)

TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.

Analysis of the Molecular Mechanism of Huangqi Herb Treating COVID- 19 with Myocardial Injury by Pharmacological Tools, Programming Software and Molecular Docking.

Huangqi with the capacity to resist virus and preserve myocardium is a potential herb for treating patients with COVID-19 and related myocardial injury. We applied network pharmacology method and programming software including R and Perl to explore the probable mechanism of Huangqi fighting against the disease. Ingredients and target gene names of Huangqi were obtained from TCMSP database. Disease-associated genes were mined by searching GeneCards database. Venny online software was applied to draw Venn diagram of intersection genes. Cytoscape software was used to set up the network of disease, drug, compounds and targets. STRING database was applied to set up protein protein interaction (PPI) network. With intersection genes imported into WEBGESALT database, gene ontology (GO) analysis was completed. An R script basing on Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to obtain KEGG pathways. Finally, we used AutoDockTools 1.5.6 software for molecular docking and PyMOL to visualize the docking details. We obtained 20 active components and 18 potential target genes to construct a network, and found out quercetin and kaempferol were core ingredients. Key targets included EGFR, MAPK8, IL6, CASP3, RELA and PPARG. Huangqi showed its potential to reduce inflammatory response to prevent cytokine storm by inhibiting EGFR, IL6 and MAPK and protect myocardium by inhibiting apoptosis and oxidant stress. Huangqi may also work by adjusting ubiquitin and regulating multiple viral pathways. Huangqi may play a therapeutic role in treating COVID-19 with myocardial injury by the effects of resisting virus and protecting myocardium concurrently.

Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction.

Previous research has suggested that vanishing white matter disease (VWMD) astrocytes fail to fully differentiate and respond differently to cellular stresses compared to healthy astrocytes. However, few studies have investigated potential VWMD therapeutics in monoculture patient-derived cell-based models. To investigate the impact of alterations in astrocyte expression and function in VWMD, astrocytes were differentiated from patient and control induced pluripotent stem cells and analyzed by proteomics, pathway analysis, and functional assays, in the absence and presence of stressors or potential therapeutics. Vanishing white matter disease astrocytes demonstrated significantly reduced expression of astrocyte markers and markers of inflammatory activation or cellular stress relative to control astrocytes. These alterations were identified both in the presence and absence of polyinosinic:polycytidylic acid stimuli, which is used to simulate viral infections. Pathway analysis highlighted differential signaling in multiple pathways in VWMD astrocytes, including eukaryotic initiation factor 2 (EIF2) signaling, oxidative stress, oxidative phosphorylation (OXPHOS), mitochondrial function, the unfolded protein response (UPR), phagosome regulation, autophagy, ER stress, tricarboxylic acid cycle (TCA) cycle, glycolysis, tRNA signaling, and senescence pathways. Since oxidative stress and mitochondrial function were two of the key pathways affected, we investigated whether two independent therapeutic strategies could ameliorate astrocyte dysfunction: edaravone treatment and mitochondrial transfer. Edaravone treatment reduced differential VWMD protein expression of the UPR, phagosome regulation, ubiquitination, autophagy, ER stress, senescence, and TCA cycle pathways. Meanwhile, mitochondrial transfer decreased VWMD differential expression of the UPR, glycolysis, calcium transport, phagosome formation, and ER stress pathways, while further modulating EIF2 signaling, tRNA signaling, TCA cycle, and OXPHOS pathways. Mitochondrial transfer also increased the gene and protein expression of the astrocyte marker, glial fibrillary acidic protein (GFAP) in VWMD astrocytes. This study provides further insight into the etiology of VWMD astrocytic failure and suggests edaravone and mitochondrial transfer as potential candidate VWMD therapeutics that can ameliorate disease pathways in astrocytes related to oxidative stress, mitochondrial dysfunction, and proteostasis.

MEN1 Degradation Induced by Neddylation and the CUL4B-DCAF7 Axis Promotes Pancreatic Neuroendocrine Tumor Progression.

Identification of neddylation and ubiquitination pathways that regulate MEN1 protein stability provides an opportunity for therapeutic interventions for treating patients with pancreatic neuroendocrine tumors.

UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2’s K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.

Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice.

Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein-protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.

BrUFO positively regulates the infection of Chinese cabbage by Plasmodiophora brassicae.

Chinese cabbage is one of the most important vegetable crops in China. However, the clubroot disease caused by the infection of Plasmodiophora brassicae (P. brassicae) has seriously affected the yield and quality of Chinese cabbage. In our previous study, BrUFO gene was found to be significantly up-regulated in diseased roots of Chinese cabbage after inoculation with P. brassicae. UFO (UNUSUAL FLORAL ORGANS) have the properties of substrate recognition during ubiquitin-mediated proteolysis. A variety of plant can activate immunity response through the ubiquitination pathway. Therefore, it is very important to study the function of UFO in response to P. brassicae. In this study, The expression pattern of BrUFO Gene was measured by qRT-PCR and In situ Hybridization (ISH). The expression location of BrUFO in cells was determined by subcellular localization. The function of BrUFO was verified by Virus-induced Gene Silencing (VIGS). proteins interacting with BrUFO protein were screened by yeast two-hybrid. Quantitative real-time polymerase chain reactions (qRT-PCR) and in situ hybridization analysis showed that expression of BrUFO gene in the resistant plants was lower than that in susceptible plants. Subcellular localization analysis showed that BrUFO gene was expressed in the nucleus. Virus-induced gene silencing (VIGS) analysis showed that silencing of BrUFO gene reduced the incidence of clubroot disease. Six proteins interacting with BrUFO protein were screened by Y2H assay. Two of them (Bra038955, a B-cell receptor-associated 31-like protein and Bra021273, a GDSL-motif esterase/acyltransferase/lipase Enzyme) were confirmed to strongly interact with BrUFO protein. BrUFO gene should be a key gene of chinese cabbage against the infection of P. brassicae. BrUFO gene silencing improves the resistance of plants to clubroot disease. BrUFO protein may interact with CUS2 to induce ubiquitination in PRR-mediated PTI reaction through GDSL lipases, so as to achieve the effect of Chinese cabbage against the infection of P. brassicae.

The mutual regulation between DcEBF1/2 and DcEIL3-1 is involved in ethylene induced petal senescence in carnation (Dianthus caryophyllus L.).

Carnation (Dianthus caryophyllus L.) is a respiratory climacteric flower, comprising one of the most important cut flowers that is extremely sensitive to plant hormone ethylene. Ethylene signaling core transcription factor DcEIL3-1 plays a key role in ethylene induced petal senescence in carnation. However, how the dose of DcEIL3-1 is regulated in the carnation petal senescence process is still not clear. Here, we screened out two EBF (EIN3 Binding F-box) genes, DcEBF1 and DcEBF2, which showed quick elevation by ethylene treatment according to the ethylene induced carnation petal senescence transcriptome. Silencing of DcEBF1 and DcEBF2 accelerated, whereas overexpression of DcEBF1 and DcEBF2 delayed, ethylene induced petal senescence in carnation by influencing DcEIL3-1 downstream target genes but not DcEIL3-1 itself. Furthermore, DcEBF1 and DcEBF2 interact with DcEIL3-1 to degrade DcEIL3-1 via an ubiquitination pathway in vitro and in vivo. Finally, DcEIL3-1 binds to the promoter regions of DcEBF1 and DcEBF2 to activate their expression. In conclusion, the present study reveals the mutual regulation between DcEBF1/2 and DcEIL3-1 during ethylene induced petal senescence in carnation, which not only expands our understanding about ethylene signal regulation network in the carnation petal senescence process, but also provides potential targets with respect to breeding a cultivar of long-lived cut carnation.

The Role of Ubiquitin-Proteasome System in the Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus-2 Disease.

Different protein degradation pathways exist in cells. However, the bulk of cellular proteins are degraded by the ubiquitin-proteasome system (UPS), which is one of these pathways. The upkeep of cellular protein homeostasis is facilitated by the ubiquitin-proteasome system, which has a variety of important functions. With the emergence of eukaryotic organisms, the relationship between ubiquitylation and proteolysis by the proteasome became apparent. Severe acute respiratory syndrome coronavirus-2 (SARS-Coronavirus-2) hijacks the ubiquitin-proteasome system and causes their viral proteins to become ubiquitinated, facilitating assembly and budding. Ubiquitination of the enzyme keratin-38 (E-K38) residue gave the virion the ability to engage with at least one putative cellular receptor, T-cell immunoglobin-mucin (TIM-1), boosting virus entry, reproduction, and pathogenesis. A fraction of infectious viral particles produced during replication have been ubiquitinated. The ubiquitin system promotes viral replication. In order to replicate their viral genome after entering the host cell, viruses combine the resources of the host cell with recently generated viral proteins. Additionally, viruses have the ability to encode deubiquitinating (DUB)-active proteins that can boost viral replication through both direct and indirect means. The SARS-Coronavirus-2 papain-like protease (PLpro) protein is a DUB enzyme that is necessary for breaking down viral polyproteins to create a working replicase complex and promote viral propagation. The ubiquitin-like molecule interferon-stimulated gene 15 (ISG15), which is likewise a regulator of the innate immune response and has antiviral characteristics, can also be broken down by this enzyme. However, limiting the E1-activating enzyme's ability to suppress the ubiquitination pathway prevented virus infection but did not prevent viral RNA genome translation. Numerous investigations have revealed that the use of proteasome inhibitors has a detrimental effect on the replication of SARS-Coronavirus-2 and other viruses in the host cell. Studies have shown that the use of proteasome inhibitors is also known to deplete free cellular ubiquitin, which may have an impact on viral replication and other vital cellular functions.

Abstract P3-05-50: Comprehensive Analysis of Pyroptotic Gene Prognostic Signatures Associated with Tumor Immune Microenvironment and Genomic Mutation in Breast Cancer

Abstract Background: Breast cancer is becoming a tumor with the highest morbidity rate, and inflammation-induced cell death namely pyroptosis reportedly plays dual roles in cancers. However, the specific mechanism between pyroptosis and the clinical prognosis of breast cancer patients is indistinct. Hence, novel pyroptosis-related biomarkers and their contributing factors deserve further exploration to predict the prognosis in breast cancer. Methods: Pearson’s correlation analysis, and univariate and multivariate Cox regression analysis were utilized to obtain six optimal pyroptosis-related gene prognostic signatures (Pyro-GPS). The risk score of each breast cancer patient was calculated. Next, a Pyro-GPS risk model was constructed and verified in TCGA cohort (n=1,089) and GSE20711 cohort (n=88). Then analyses of immune microenvironment, genomic variation, functional enrichment, drug response and clinicopathologic feature stratification associated with the risk score of Pyro-GPS were performed. Ultimately, a nomogram based on the risk score and several significant clinicopathologic features was established. Results: The low-risk breast cancer patients have better survival outcomes than the high-risk patients. The low-risk patients also show higher immune cell infiltration levels and immune-oncology target expression levels. There is no significant difference in genetic variation between the two risk groups, but the frequency of gene mutations varies. Functional enrichment analysis shows that the low-risk patients are prominently correlated with immune-related pathways, whereas the high-risk patients are enriched in cell cycle, ubiquitination, mismatch repair, homologous recombination and biosynthesis-related pathways. Pyro-GPS is positively correlated with the IC50 of anti-tumor drugs. Furthermore, comprehensive analyses based on risk score and clinicopathological features were performed to predict the prognosis of breast cancer patients. Conclusions: The risk score of Pyro-GPS can serve as a promising hallmark to predict the prognosis of BRCA patients. Risk score evaluation may assist to acquire relevant information of tumor immune microenvironment, genomic mutation status, functional pathways and drug sensitivity, and thus provide more effective personalized strategies. Citation Format: Hongfei Zhang, Xiafei Yu, Junzhe Yang, Xiaoan Liu, Yanhui Zhu. Comprehensive Analysis of Pyroptotic Gene Prognostic Signatures Associated with Tumor Immune Microenvironment and Genomic Mutation in Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-50.

Physiological and transcriptomic responses to cold waves of the most cold-tolerant mangrove, Kandelia obovata.

Mangrove forests inhabit tropical or subtropical intertidal zones and have remarkable abilities in coastline protection. Kandelia obovata is considered the most cold-tolerant mangrove species and has been widely transplanted to the north subtropical zone of China for ecological restoration. However, the physiological and molecular mechanisms of K. obovata under colder climate was still unclear. Here, we manipulated the typical climate of cold waves in the north subtropical zone with cycles of cold/recovery and analyzed the physiological and transcriptomic responses of seedlings. We found that both physiological traits and gene expression profiles differed between the first and later cold waves, indicating K. obovata seedlings were acclimated by the first cold experience and prepared for latter cold waves. 1,135 cold acclimation-related genes (CARGs) were revealed, related to calcium signaling, cell wall modification, and post-translational modifications of ubiquitination pathways. We identified the roles of CBFs and CBF-independent transcription factors (ZATs and CZF1s) in regulating the expression of CARGs, suggesting both CBF-dependent and CBF- independent pathways functioned in the cold acclimation of K. obovata. Finally, we proposed a molecular mechanism of K. obovata cold acclimation with several key CARGs and transcriptional factors involved. Our experiments reveal strategies of K. obovata coping with cold environments and provide prospects for mangrove rehabilitation and management.

The F-box protein UFO controls flower development by redirecting the master transcription factor LEAFY to new cis-elements.

In angiosperms, flower development requires the combined action of the transcription factor LEAFY (LFY) and the ubiquitin ligase adaptor F-box protein, UNUSUAL FLORAL ORGANS (UFO), but the molecular mechanism underlying this synergy has remained unknown. Here we show in transient assays and stable transgenic plants that the connection to ubiquitination pathways suggested by the UFO F-box domain is mostly dispensable. On the basis of biochemical and genome-wide studies, we establish that UFO instead acts by forming an active transcriptional complex with LFY at newly discovered regulatory elements. Structural characterization of the LFY-UFO-DNA complex by cryo-electron microscopy further demonstrates that UFO performs this function by directly interacting with both LFY and DNA. Finally, we propose that this complex might have a deep evolutionary origin, largely predating flowering plants. This work reveals a unique mechanism of an F-box protein directly modulating the DNA binding specificity of a master transcription factor.

Genome-Wide Identification, Characterization, and Expression Analysis of the U-Box Gene Family in Punica granatum L.

The ubiquitination pathway is essential for several developmental phases in plants, and the U-box protein family of ubiquitin ligases plays an important role in this process. However, little is known about pomegranate’s PUB genes. In this investigation, the pomegranate U-box gene family was identified using whole-genome sequencing data. We identified a total of 56 members of the pomegranate U-box family based on the U-box domain, and the PgPUBs were classified into four groups. Chromosomal localization, phylogenetic analysis, motif distribution, gene duplications, cis-acting elements, and expression profiling were also investigated. The PgPUB genes were unevenly distributed among the eight pomegranate chromosomes, and collinear duplicated genes were identified between the Arabidopsis thaliana genome and the Punica granatum genome. Furthermore, the gene expression analysis revealed that expression of U-box genes in pomegranate was induced by abiotic stressors. Collectively, our findings provide insight into the U-box gene family and will assist in understanding the functional divergence of U-box genes in Punica granatum L.