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Disease Models & Mechanisms Jun 2024Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in...
Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9 simulating a CMS patient. As Gfpt1 exon 9 is exclusively included in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (i) UDP-HexNAc, CMP-NeuAc, and protein O-GlcNAcylations were reduced in skeletal muscles; (ii) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (iii) markers for unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of ER stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation, and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.
PubMed: 38903011
DOI: 10.1242/dmm.050768 -
Stem Cell Research & Therapy Jun 2024Telomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by...
BACKGROUND
Telomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by alternative telomere lengthening of telomeres (ALT) through recombination-based mechanisms. Additional mechanisms that may regulate telomere maintenance remain to be explored. Simultaneous measurement of telomere length and transcriptome in the same human embryonic stem cell (hESC) revealed that mRNA expression levels of UBQLN1 exhibit linear relationship with telomere length.
METHODS
In this study, we first generated UBQLN1-deficient hESCs and compared with the wild-type (WT) hESCs the telomere length and molecular change at RNA and protein level by RNA-seq and proteomics. Then we identified the potential interacting proteins with UBQLN1 using immunoprecipitation-mass spectrometry (IP-MS). Furthermore, the potential mechanisms underlying the shortened telomeres in UBQLN1-deficient hESCs were analyzed.
RESULTS
We show that Ubiquilin1 (UBQLN1) is critical for telomere maintenance in human embryonic stem cells (hESCs) via promoting mitochondrial function. UBQLN1 deficiency leads to oxidative stress, loss of proteostasis, mitochondria dysfunction, DNA damage, and telomere attrition. Reducing oxidative damage and promoting mitochondria function by culture under hypoxia condition or supplementation with N-acetylcysteine partly attenuate the telomere attrition induced by UBQLN1 deficiency. Moreover, UBQLN1 deficiency/telomere shortening downregulates genes for neuro-ectoderm lineage differentiation.
CONCLUSIONS
Altogether, UBQLN1 functions to scavenge ubiquitinated proteins, preventing their overloading mitochondria and elevated mitophagy. UBQLN1 maintains mitochondria and telomeres by regulating proteostasis and plays critical role in neuro-ectoderm differentiation.
Topics: Humans; Human Embryonic Stem Cells; Autophagy-Related Proteins; Mitochondria; Proteostasis; Telomere; Telomere Homeostasis; Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Oxidative Stress; DNA Damage
PubMed: 38902824
DOI: 10.1186/s13287-024-03789-y -
Cell Death & Disease Jun 2024Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome...
Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics.
Topics: Ferroptosis; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Animals; Proteasome Endopeptidase Complex; Ubiquitin; Mice; Amino Acid Transport System y+; Oncogene Proteins; Membrane Proteins; Cell Line, Tumor; Ubiquitination; Mice, Nude; Proteolysis
PubMed: 38902268
DOI: 10.1038/s41419-024-06836-x -
PLoS Pathogens Jun 2024Coronavirus (CoV) nonstructural protein 1 (nsp1) is considered a pathogenic factor due to its ability to inhibit host antiviral responses by inducing general shutoff of...
Coronavirus (CoV) nonstructural protein 1 (nsp1) is considered a pathogenic factor due to its ability to inhibit host antiviral responses by inducing general shutoff of host protein synthesis. Nsp1 is expressed by α- and β-CoVs, but its functions and strategies to induce host shutoff are not fully elucidated. We compared the nsp1s from two β-CoVs (SARS-CoV and SARS-CoV-2) and two α-CoVs (NL63 and 229E) and found that NL63 nsp1 has the strongest shutoff activity. Unlike SARS-CoV nsp1s, which bind to 40S ribosomes and block translation of cellular mRNA, NL63 nsp1 did not inhibit translation of mRNAs transfected into cells. Instead, NL63 nsp1 localized to the nucleus and specifically inhibited transcription of genes under an RNA polymerase II (RNAPII) promoter. Further analysis revealed that NL63 nsp1 induces degradation of the largest subunit of RNAPII, Rpb1. This degradation was detected regardless of the phosphorylation state of Rpb1 and was blocked by the proteasome inhibitor MG132. We also found that Rpb1 was ubiquitinated in NL63-infected cells, and inhibition of ubiquitination by a ubiquitin activating enzyme inhibitor (TAK243) prevented degradation of Rpb1 in virus-infected cells. These data reveal an unrecognized strategy of host shutoff by human α-CoV NL63: targeting host transcription by inducing Rpb1 degradation to prevent host protein expression. Our study indicates that viruses within the same family can use completely distinct mechanisms to regulate host antiviral responses.
PubMed: 38900816
DOI: 10.1371/journal.ppat.1012329 -
ELife Jun 2024The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling...
E3 ubiquitin ligase Deltex facilitates the expansion of Wingless gradient and antagonizes Wingless signaling through a conserved mechanism of transcriptional effector Armadillo/β-catenin degradation.
The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling is associated with a wide range of pathologies in humans. Tight regulation of Wnt/Wg signaling is required to maintain proper cellular homeostasis. Here, we report a novel role of E3 ubiquitin ligase Deltex in Wg signaling regulation. genetically interacts with nd its pathway components. Furthermore, Dx LOF results in a reduced spreading of Wg while its over-expression expands the diffusion gradient of the morphogen. We attribute this change in Wg gradient to the endocytosis of Wg through Dx which directly affects the short- and long-range Wg targets. We also demonstrate the role of Dx in regulating Wg effector Armadillo where Dx down-regulates Arm through proteasomal degradation. We also showed the conservation of Dx function in the mammalian system where DTX1 is shown to bind with β-catenin and facilitates its proteolytic degradation, spotlighting a novel step that potentially modulates Wnt/Wg signaling cascade.
Topics: Animals; Wnt1 Protein; Drosophila Proteins; Ubiquitin-Protein Ligases; Armadillo Domain Proteins; Proteolysis; Wnt Signaling Pathway; beta Catenin; Drosophila melanogaster; Signal Transduction; Humans; Drosophila; Transcription Factors
PubMed: 38900140
DOI: 10.7554/eLife.88466 -
ELife Jun 2024The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn)...
The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1,200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble aSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.
PubMed: 38899618
DOI: 10.7554/eLife.98649 -
Haematologica Jun 2024Hematological cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the...
Hematological cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding was demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs-inducing actin polymerization.
PubMed: 38899342
DOI: 10.3324/haematol.2022.282672 -
Kidney International Reports Jun 2024Current therapeutic management of lupus nephritis (LN) fails to induce long-term remission in over 50% of patients, highlighting the urgent need for additional options.
INTRODUCTION
Current therapeutic management of lupus nephritis (LN) fails to induce long-term remission in over 50% of patients, highlighting the urgent need for additional options.
METHODS
We analyzed differentially expressed genes (DEGs) in peripheral blood from patients with active LN ( = 41) and active nonrenal lupus ( = 62) versus healthy controls (HCs) ( = 497) from the European PRECISESADS project (NTC02890121), and dysregulated gene modules in a discovery ( = 26) and a replication ( = 15) set of active LN cases.
RESULTS
Replicated gene modules qualified for correlation analyses with serologic markers, and regulatory network and druggability analysis. Unsupervised coexpression network analysis revealed 20 dysregulated gene modules and stratified the active LN population into 3 distinct subgroups. These subgroups were characterized by low, intermediate, and high interferon (IFN) signatures, with differential dysregulation of the "B cell" and "plasma cells/Ig" modules. Drugs annotated to the IFN network included CC-motif chemokine receptor 1 (CCR1) inhibitors, programmed death-ligand 1 (PD-L1) inhibitors, and irinotecan; whereas the anti-CD38 daratumumab and proteasome inhibitor bortezomib showed potential for counteracting the "plasma cells/Ig" signature. analysis demonstrated the low-IFN subgroup to benefit from calcineurin inhibition and the intermediate-IFN subgroup from B-cell targeted therapies. High-IFN patients exhibited greater anticipated response to anifrolumab whereas daratumumab appeared beneficial to the intermediate-IFN and high-IFN subgroups.
CONCLUSION
IFN upregulation and B and plasma cell gene dysregulation patterns revealed 3 subgroups of LN, which may not necessarily represent distinct disease phenotypes but rather phases of the inflammatory processes during a renal flare, providing a conceptual framework for precision medicine in LN.
PubMed: 38899167
DOI: 10.1016/j.ekir.2024.03.014 -
Cell Communication and Signaling : CCS Jun 2024Endocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of...
BACKGROUND
Endocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors.
METHODS
Bioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth.
RESULTS
RACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa.
CONCLUSION
In summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.
Topics: Male; Humans; Drug Resistance, Neoplasm; Receptors, Androgen; GTPase-Activating Proteins; Prostatic Neoplasms; Cell Line, Tumor; Animals; Proto-Oncogene Mas; Gene Expression Regulation, Neoplastic; Phenylthiohydantoin; Mice, Nude; Nitriles; Mice; Benzamides; Cell Proliferation; Proto-Oncogene Proteins c-mdm2; Signal Transduction
PubMed: 38898473
DOI: 10.1186/s12964-024-01703-w -
BMC Medical Genomics Jun 2024Immunoregulatory drugs regulate the ubiquitin-proteasome system, which is the main treatment for multiple myeloma (MM) at present. In this study, bioinformatics analysis...
BACKGROUND
Immunoregulatory drugs regulate the ubiquitin-proteasome system, which is the main treatment for multiple myeloma (MM) at present. In this study, bioinformatics analysis was used to construct the risk model and evaluate the prognostic value of ubiquitination-related genes in MM.
METHODS AND RESULTS
The data on ubiquitination-related genes and MM samples were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The consistent cluster analysis and ESTIMATE algorithm were used to create distinct clusters. The MM prognostic risk model was constructed through single-factor and multiple-factor analysis. The ROC curve was plotted to compare the survival difference between high- and low-risk groups. The nomogram was used to validate the predictive capability of the risk model. A total of 87 ubiquitination-related genes were obtained, with 47 genes showing high expression in the MM group. According to the consistent cluster analysis, 4 clusters were determined. The immune infiltration, survival, and prognosis differed significantly among the 4 clusters. The tumor purity was higher in clusters 1 and 3 than in clusters 2 and 4, while the immune score and stromal score were lower in clusters 1 and 3. The proportion of B cells memory, plasma cells, and T cells CD4 naïve was the lowest in cluster 4. The model genes KLHL24, HERC6, USP3, TNIP1, and CISH were highly expressed in the high-risk group. AICAr and BMS.754,807 exhibited higher drug sensitivity in the low-risk group, whereas Bleomycin showed higher drug sensitivity in the high-risk group. The nomogram of the risk model demonstrated good efficacy in predicting the survival of MM patients using TCGA and GEO datasets.
CONCLUSIONS
The risk model constructed by ubiquitination-related genes can be effectively used to predict the prognosis of MM patients. KLHL24, HERC6, USP3, TNIP1, and CISH genes in MM warrant further investigation as therapeutic targets and to combat drug resistance.
Topics: Humans; Multiple Myeloma; Computational Biology; Prognosis; Ubiquitination; Gene Expression Regulation, Neoplastic; Biomarkers, Tumor; Nomograms; Cluster Analysis
PubMed: 38898455
DOI: 10.1186/s12920-024-01937-0