Ubiquitination Targets Research Articles

Nonphototrophic hypocotyl 3 domain proteins: traffic directors, hitchhikers, or both?

The nonphototrophic hypocotyl 3 (NPH3) domain is plant specific and of unknown function. It is nearly always attached to an N-terminal BTB domain and a largely unstructured C-terminal region. Recent reports revealed NPH3-domain GTPase activity and connection to intracellular trafficking, condensate formation, membrane attachment of the C-terminal region for some NPH3-domain proteins and, at the physiological level, drought-related function for at least one NPH3-domain protein. We integrate these new ideas of NPH3-domain protein function into two, nonexclusive, working models: the 'traffic director' model, whereby NPH3-domain proteins regulate intracellular trafficking and, the 'hitchhiker' model whereby NPH3-domain proteins ride the trafficking system to find ubiquitination targets. Determining which model best applies to uncharacterized NPH3-domain proteins will contribute to understanding intracellular trafficking and environmental responses.

USP8 promotes intracellular infection by enhancing ESCRT-mediated membrane repair, limiting xenophagy, and reducing oxidative stress

ABSTRACT The host ESCRT-machinery repairs damaged endolysosomal membranes. If damage persists, selective macroautophagy/autophagy clears the damaged compartment. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that damages the phagosomal membrane and targets ESCRT-mediated repair as part of its virulence program. The E3 ubiquitin ligases PRKN and SMURF1 promote autophagic capture of damaged, Mtb-containing phagosomes. Because ubiquitination is a reversible process, we anticipated that host deubiquitinases (DUBs) would also be involved. Here, we screened all predicted mouse DUBs for their role in ubiquitin targeting and control of intracellular Mtb. We show that USP8 (ubiquitin specific peptidase 8) colocalizes with intracellular Mtb, recognizes phagosomal membrane damage, and is required for ESCRT-dependent membrane repair. Furthermore, we show that USP8 regulates the NFE2L2/NRF2-dependent antioxidant signature. Taken together, our study demonstrates a central role of USP8 in promoting Mtb intracellular growth by promoting phagosomal membrane repair, limiting ubiquitin-driven selective autophagy, and reducing oxidative stress. Abbreviation: BMDMs: bone marrow-derived macrophages; CFUs: colony-forming units; DUB: deubiquitinase; ESCRT: endosomal sorting complexes required for transport; LLOMe: L-leucyl-L-leucine methyl ester; MFI: mean fluorescence intensity; MOI: multiplicity of infection; Mtb: Mycobacterium tuberculosis; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; PMA: phorbol 12-myristate 13-acetate; ROS: reactive oxygen species; USP8: ubiquitin specific peptidase 8

Iron-sulfur cluster loss in mitochondrial CISD1 mediates PINK1 loss-of-function phenotypes.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila orthologue of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.

C646 degrades Exportin-1 to modulate p300 chromatin occupancy and function

Molecular glues can induce proximity between a target protein and ubiquitin ligases to induce target degradation, but strategies for their discovery remain limited. We screened 3,200 bioactive small molecules and identified that C646 requires neddylation-dependent protein degradation to induce cytotoxicity. Although the histone acetyltransferase p300 is the canonical target of C646, we provide extensive evidence that C646 directly targets and degrades Exportin-1 (XPO1). Multiple cellular phenotypes induced by C646 were abrogated in cells expressing the known XPO1C528S drug-resistance allele. While XPO1 catalyzes nuclear-to-cytoplasmic transport of many cargo proteins, it also directly binds chromatin. We demonstrate that p300 and XPO1 co-occupy hundreds of chromatin loci. Degrading XPO1 using C646 or the known XPO1 modulator S109 diminishes the chromatin occupancy of both XPO1 and p300, enabling direct targeting of XPO1 to phenocopy p300 inhibition. This work highlights the utility of drug-resistant alleles and further validates XPO1 as a targetable regulator of chromatin state.

A small-molecule degron with a phenylpropionic acid scaffold

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin–proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

SCFFBXW5-mediated degradation of AQP3 suppresses autophagic cell death through the PDPK1-AKT-MTOR axis in hepatocellular carcinoma cells

ABSTRACT AQP3 (aquaporin 3 (Gill blood group)), a member of the AQP family, is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Beyond its role in fluid transport, AQP3 plays a significant role in regulating various aspects of tumor cell behavior, including cell proliferation, migration, and invasion. Nevertheless, the underlying regulatory mechanism of AQP3 in tumors remains unclear. Here, for the first time, we report that AQP3 is a direct target for ubiquitination by the SCFFBXW5 complex. In addition, we revealed that downregulation of FBXW5 significantly induced AQP3 expression to prompt macroautophagic/autophagic cell death in hepatocellular carcinoma (HCC) cells. Mechanistically, AQP3 accumulation induced by FBXW5 knockdown led to the degradation of PDPK1/PDK1 in a lysosomal-dependent manner, thus inactivating the AKT-MTOR pathway and inducing autophagic death in HCC. Taken together, our findings revealed a previously undiscovered regulatory mechanism through which FBXW5 degraded AQP3 to suppress autophagic cell death via the PDPK1-AKT-MTOR axis in HCC cells. Abbreviation: BafA1: bafilomycin A1; CQ: chloroquine; CRL: CUL-Ring E3 ubiquitin ligases; FBXW5: F-box and WD repeat domain containing 5; HCC: hepatocellular carcinoma; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; 3-MA: 3-methyladenine; PDPK1/PDK1: 3-phosphoinositide dependent protein kinase 1; RBX1/ROC1: ring-box 1; SKP1: S-phase kinase associated protein 1; SCF: SKP1-CUL1-F-box protein.

The E3 Ubiquitin Protein Ligase LINCR Amplifies the TLR-Mediated Signals through Direct Degradation of MKP1.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

PDX proteins from Arabidopsis thaliana as novel substrates of cathepsin B: implications for vitamin B6 biosynthesis regulation.

Vitamin B6 is a critical molecule for metabolism, development, and stress sensitivity in plants. It is a cofactor for numerous biochemical reactions, can serve as an antioxidant, and has the potential to increase tolerance against both biotic and abiotic stressors. Due to the importance of vitamin B6, its biosynthesis is likely tightly regulated. Plants can synthesize vitamin B6 de novo via the concerted activity of Pyridoxine Biosynthesis Protein 1 (PDX1) and PDX2. Previously, PDX proteins have been identified as targets for ubiquitination, indicating they could be marked for degradation by two highly conserved pathways: the Ubiquitin Proteasome Pathway (UPP) and the autophagy pathway. Initial experiments show that PDXs are in fact degraded, but surprisingly, in a ubiquitin-independent manner. Inhibitor studies pointed toward cathepsin B, a conserved lysosomal cysteine protease, which is implicated in both programed cell death and autophagy in humans and plants. In plants, cathepsin Bs are poorly described, and no confirmed substrates have been identified. Here, we present PDX proteins from Arabidopsis thaliana as interactors and substrates of a plant Cathepsin B. These findings not only describe a novel cathepsin B substrate in plants, but also provide new insights into how plants regulate de novo biosynthesis of vitamin B6.

Abstract IA016: Molecular diversity of uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer with a high recurrence rate and poor prognosis disproportionately affecting black women. Whole-exome-sequencing analysis revealed that up to 10% of USC harbor features of ultra/hypermutation due to a deficiency of polymerase e (POLE) and/or mismatched-repair genes (MMRd). These tumors are notable for having a high number of single nucleotide variants (SNV), no LOH segments or copy-number variants (CNV) and a peculiar genetic signature characterized by an increased rate of C > A transversions. In contrast, the overwhelming majority of USC (~ 90%) are POLE/MMR proficient, have a low number of SNV and demonstrate alteration in genes including TP53, PIK3CA mutation/amplification, ERBB2 amplification, CCNE1 amplification, FBXW7 mutation/deletion, PPP2R1A mutation, and somatic mutations involving homologous recombination genes. Frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling-complex, and TAF1, an element of the core TFIID transcriptional machinery are also detected in a significant number of USC. Somatic CNVs, a sign of genomic instability, are identified in the majority of USC with multiple copy number gains and copy number losses, with the most frequent events taking place in pathways similar to those found from analysis of SNVs. In addition to loss of TP53, frequent deletion of a small segment of chromosome 19 containing MBD3, a member of the NuRD-chromatin-modification-complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (encoding for the HER2/neu receptor) and CCNE1 (a target of FBXW7-mediated ubiquitination) are commonly detected. Overall, these findings suggest that both somatic point mutations (SNV) and somatic CNV play a major role in the pathogenesis of USC. Importantly, the frequent mutation of DNA damage, chromatin remodeling, cell cycle and cell proliferation pathways in USC including the overexpression/amplification of HER2/neu, CCNE1 and the tumor-associated calcium signal transducer 2 (TACSTD2/TROP2) genes, suggest novel targets for therapy against this lethal variant of endometrial cancer. Citation Format: Alessandro D. Santin. Molecular diversity of uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA016.

Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

Interaction between Gtr2p and ribosomal Rps31p affects the incorporation of Rps31p into ribosomes of Saccharomyces cerevisiae

In yeast, ras-like small G proteins, Gtr1p and Gtr2p, form heterodimers that affect cell division, detect amino acids, and regulate the activity of TORC1, a protein complex that integrates various signals, including those related to nutrient availability, growth factors, and stress signals. To explore novel roles of Gtr2p, yeast two-hybrid screening was performed using gtr2S23Np, an active form of Gtr2p, which identified Rps31p and Rpl12p as Gtr2p-interacting proteins. In the present study, we found that Gtr2p, but not Gtr1p, interacts with Rps31p, a 40S ribosomal subunit, and a component of the ubiquitin fusion protein Ubi3p, which is essential for the initiation and elongation of translation. In yeast cells expressing gtr2Q66Lp, an inactive form of Gtr2p, the interaction between Rps31p and gtr2Q66Lp, as well as the level of exogenous expression of Rps31p, was reduced. However, the level of exogenous expression of Rpl12p was unaffected. Introducing a mutation in ubiquitin target lysine residues to arginine (rps31-K5R) restored the level of exogenously expressed Rps31p and rescued the rapamycin and caffeine sensitivity of gtr2Q66L cells. Sucrose density gradient centrifugation of yeast cell lysate expressing Rps31p and gtr2Q66Lp revealed that exogenously expressed Rps31p was poorly incorporated, whereas rps31-K5Rp was efficiently incorporated, into ribosomes. These results suggest that Gtr2p influences incorporation of Rps31p into ribosomes and contributes to drug resistance through its interaction with Rps31p.

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCFβ-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organizing protein Dbn1 as a major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCFβ-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized β-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA damage-dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX’s ability to identify targets of stimulus-regulated ubiquitylation and reveal an SCFβ-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

GW9 determines grain size and floral organ identity in rice.

Grain weight is an important determinant of grain yield. However, the underlying regulatory mechanisms for grain size remain to be fully elucidated. Here, we identify a rice mutant grain weight 9 (gw9), which exhibits larger and heavier grains due to excessive cell proliferation and expansion in spikelet hull. GW9 encodes a nucleus-localized protein containing both C2H2 zincfinger (C2H2-ZnF) and VRN2-EMF2-FIS2-SUZ12 (VEFS) domains, serving as a negative regulator of grain size and weight. Interestingly, the non-frameshift mutations in C2H2-ZnF domain result in increased plant height and larger grain size, whereas frameshift mutations in both C2H2-ZnF and VEFS domains lead to dwarf and malformed spikelet. These observations indicated the dual functions of GW9 in regulating grain size and floral organ identity through the C2H2-ZnF and VEFS domains, respectively. Further investigation revealed the interaction between GW9 and the E3 ubiquitin ligase protein GW2, with GW9 being the target of ubiquitination by GW2. Genetic analyses suggest that GW9 and GW2 function in a coordinated pathway controlling grain size and weight. Our findings provide a novel insight into the functional role of GW9 in the regulation of grain size and weight, offering potential molecular strategies for improving rice yield.

Delivering on Cell-Selective Protein Degradation Using Chemically Tailored PROTACs.

PROTACs (Proteolysis-Targeting Chimeras) have emerged as a groundbreaking class of chemical tools that facilitate the degradation of target proteins by leveraging the ubiquitin-proteasome system (UPS). However, the effective utilization of PROTACs in chemical biology studies and therapeutics encounters significant challenges when it comes to achieving cell-selective protein degradation and in vivo applications. This review article aims to shed light on recent advancements in the development of Pro-PROTACs, which exhibit controlled protein degradation capabilities in response to external stimuli or disease-related endogenous biochemical signals. The article delves into the specific chemical strategies employed to regulate the interaction between PROTACs and E3 ubiquitin ligases or target proteins. These strategies enable spatial and temporal control over the protein degradation potential of Pro-PROTACs. Furthermore, the review summarizes recent investigations regarding the delivery of PROTACs using biodegradable nanoparticles for in vivo applications and targeted protein degradation. Such delivery systems hold great promise for enabling efficient and selective protein degradation in vivo. Lastly, the article provides a perspective on the future design of multifunctional PROTACs and their intracellular delivery mechanisms, with a particular focus on achieving cell-selective protein degradation.

F-box only protein 2 exacerbates non-alcoholic fatty liver disease by targeting the hydroxyl CoA dehydrogenase alpha subunit.

Non-alcoholic fatty liver disease (NAFLD) is a major health burden with an increasing global incidence. Unfortunately, the unavailability of knowledge underlying NAFLD pathogenesis inhibits effective preventive and therapeutic measures. To explore the molecular mechanism of NAFLD. Whole genome sequencing (WGS) analysis was performed on liver tissues from patients with NAFLD (n = 6) and patients with normal metabolic conditions (n = 6) to identify the target genes. A NAFLD C57BL6/J mouse model induced by 16 wk of high-fat diet feeding and a hepatocyte-specific F-box only protein 2 (FBXO2) overexpression mouse model were used for in vivo studies. Plasmid transfection, co-immunoprecipitation-based mass spectrometry assays, and ubiquitination in HepG2 cells and HEK293T cells were used for in vitro studies. A total of 30982 genes were detected in WGS analysis, with 649 up-regulated and 178 down-regulated. Expression of FBXO2, an E3 ligase, was upregulated in the liver tissues of patients with NAFLD. Hepatocyte-specific FBXO2 overexpression facilitated NAFLD-associated phenotypes in mice. Overexpression of FBXO2 aggravated odium oleate (OA)-induced lipid accumulation in HepG2 cells, resulting in an abnormal expression of genes related to lipid metabolism, such as fatty acid synthase, peroxisome proliferator-activated receptor alpha, and so on. In contrast, knocking down FBXO2 in HepG2 cells significantly alleviated the OA-induced lipid accumulation and aberrant expression of lipid metabolism genes. The hydroxyl CoA dehydrogenase alpha subunit (HADHA), a protein involved in oxidative stress, was a target of FBXO2-mediated ubiquitination. FBXO2 directly bound to HADHA and facilitated its proteasomal degradation in HepG2 and HEK293T cells. Supplementation with HADHA alleviated lipid accumulation caused by FBXO2 overexpression in HepG2 cells. FBXO2 exacerbates lipid accumulation by targeting HADHA and is a potential therapeutic target for NAFLD.

Targeted degradation of MERTK and other TAM receptor paralogs by heterobifunctional targeted protein degraders.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

USP15-USP7 Axis and UBE2T Differential Expression May Predict Pathogenesis and Poor Prognosis in De Novo Myelodysplastic Neoplasm.

The aim of this study was to evaluate the expression of USP7, USP15, UBE2O, and UBE2T genes in Myelodysplastic neoplasm (MDS) to identify possible targets of ubiquitination and deubiquitination in MDS pathobiology. To achieve this, eight datasets from the Gene Expression Omnibus (GEO) database were integrated, and the expression relationship of these genes was analyzed in 1092 MDS patients and healthy controls. Our results showed that UBE2O, UBE2T, and USP7 were upregulated in MDS patients compared with healthy individuals, but only in mononucleated cells collected from bone marrow samples (p < 0.001). In contrast, only the USP15 gene showed a downregulated expression compared with healthy individuals (p = 0.03). Additionally, the upregulation of UBE2T expression was identified in MDS patients with chromosomal abnormalities compared with patients with normal karyotypes (p = 0.0321), and the downregulation of UBE2T expression was associated with MDS hypoplastic patients (p = 0.033). Finally, the USP7 and USP15 genes were strongly correlated with MDS (r = 0.82; r2 = 0.67; p < 0.0001). These findings suggest that the differential expression of the USP15-USP7 axis and UBE2T may play an important role in controlling genomic instability and the chromosomal abnormalities that are a striking characteristic of MDS.

Ubiquitin proteomics identifies RNA polymerase I as a target of the Smc5/6 complex

Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.

Identification of Ndfip1 as a novel negative regulator for spatial memory formation associated with increased ubiquitination of Beclin 1 and PTEN.

Long-term memory formation requires de novo RNA and protein synthesis. By using the differential display-polymerase chain reaction strategy, we have presently identified the Nedd4 family interacting protein 1 (Ndfip1) cDNA fragment that is differentially expressed between the slow learners and the fast learners from the water maze learning task in rats. Further, the fast learners show decreased Ndfip1 mRNA and protein expression levels than the slow learners. Spatial training similarly decreases the Ndfip1 mRNA and protein expression levels. Conversely, the Ndfip1 conditional heterozygous (cHet) mice show enhanced spatial memory performance compared to the Ndfip1flox/WT control mice. Result from co-immunoprecipitation experiment indicates that spatial training decreases the association between Ndfip1 and the E3 ubiquitin ligase Nedd4 (Nedd4-1), and we have shown that both Beclin 1 and PTEN are endogenous ubiquitination targets of Nedd4 in the hippocampus. Further, spatial training decreases endogenous Beclin 1 and PTEN ubiquitination, and increases Beclin 1 and PTEN expression in the hippocampus. On the other hand, the Becn1 conditional knockout (cKO) mice and the Pten cKO mice both show impaired spatial learning and memory performance. Moreover, the expression level of Beclin 1 and PTEN is higher in the Ndfip1 cHet mice compared with the Ndfip1flox/WT control mice. Here, we have identified Ndfip1 as a candidate novel negative regulation for spatial memory formation and this is associated with increased ubiquitination of Beclin 1 and PTEN in the hippocampus.