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Proceedings of the National Academy of... May 2024The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including , the causative agent of the...
The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including , the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.
Topics: Plasmodium falciparum; Ubiquitin Thiolesterase; Humans; Peptides; Protozoan Proteins; Antimalarials; Ubiquitin; Malaria, Falciparum
PubMed: 38739798
DOI: 10.1073/pnas.2322923121 -
MedComm May 2024The proteasome inhibitor bortezomib (BTZ) is the first-line therapy for multiple myeloma (MM). BTZ resistance largely limits its clinical application in MM....
Interleukin-33 increases the sensitivity of multiple myeloma cells to the proteasome inhibitor bortezomib through reactive oxygen species-mediated inhibition of nuclear factor kappa-B signal and stemness properties.
The proteasome inhibitor bortezomib (BTZ) is the first-line therapy for multiple myeloma (MM). BTZ resistance largely limits its clinical application in MM. Interleukin-33 (IL-33) exerts antitumor effects through various mechanisms, including enhancing antitumor immunity and promoting the apoptosis of cancer cells. Here, the synergistic anti-MM effect of IL-33 and BTZ was verified, and the underlying mechanisms were elucidated. Bioinformatic analysis indicated that IL-33 expression levels were downregulated in MM, and that BTZ-treated MM patients with high IL-33 levels had better prognosis than those with low IL-33 levels. Moreover, the patients with high IL-33 levels had a better treatment response to BTZ. Further immune analysis suggested that IL-33 can enhance the anti-MM immunity. IL-33 and BTZ synergistically inhibited proliferation and induced apoptosis of MM cells, which was mediated by the excessive accumulation of cellular reactive oxygen species (ROS). Furthermore, increased ROS hindered the nuclear translocation of NF-κB-p65, thereby decreasing the transcription of target stemness-related genes (, , and ). These effects induced by the combination therapy could be reversed by eliminating ROS by -acetylcysteine. In conclusion, our results indicated that IL-33 enhanced the sensitivity of MM to BTZ through ROS-mediated inhibition of nuclear factor kappa-B (NF-κB) signal and stemness properties.
PubMed: 38737470
DOI: 10.1002/mco2.562 -
Biology Direct May 2024Clear cell renal cell carcinoma (RCC) is the most common kidney tumor. The analysis from medical database showed that Scm-like with four MBT domains protein 2 (SFMBT2)...
BACKGROUND
Clear cell renal cell carcinoma (RCC) is the most common kidney tumor. The analysis from medical database showed that Scm-like with four MBT domains protein 2 (SFMBT2) was decreased in advanced clear cell RCC cases, and its downregulation was associated with the poor prognosis. This study aims to investigate the role of SFMBT2 in clear cell RCC.
METHODS
The expression of SFMBT2 in clear cell RCC specimens were determined by immunohistochemistry staining and western blot. The overexpression and knockdown of SFMBT2 was realized by infection of lentivirus loaded with SFMBT2 coding sequence or silencing fragment in 786-O and 769-P cells, and its effects on proliferation and metastasis were assessed by MTT, colony formation, flow cytometry, wound healing, transwell assay, xenograft and metastasis experiments in nude mice. The interaction of SFMBT2 with histone deacetylase 3 (HDAC3) and seven in absentia homolog 1 (SIAH1) was confirmed by co-immunoprecipitation.
RESULTS
In our study, SFMBT2 exhibited lower expression in clear cell RCC specimens with advanced stages than those with early stages. Overexpression of SFMBT2 inhibited the growth and metastasis of clear cell RCC cells, 786-O and 769-P, in vitro and in vivo, and its silencing displayed opposites effects. HDAC3 led to deacetylation of SFMBT2, and the HDAC3 inhibitor-induced acetylation prevented SFMBT2 from SIAH1-mediated ubiquitination modification and proteasome degradation. K687 in SFMBT2 protein molecule may be the key site for acetylation and ubiquitination.
CONCLUSIONS
SFMBT2 exerted an anti-tumor role in clear cell RCC cells, and HDAC3-mediated deacetylation promoted SIAH1-controlled ubiquitination of SFMBT2. SFMBT2 may be considered as a novel clinical diagnostic marker and/or therapeutic target of clear cell RCC, and crosstalk between its post-translational modifications may provide novel insights for agent development.
Topics: Carcinoma, Renal Cell; Humans; Ubiquitination; Acetylation; Kidney Neoplasms; Animals; Mice; Cell Line, Tumor; Mice, Nude; Cell Proliferation; Histone Deacetylases; Gene Expression Regulation, Neoplastic
PubMed: 38734627
DOI: 10.1186/s13062-024-00480-3 -
International Journal of Molecular... Apr 2024The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin....
The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.
Topics: Myocytes, Cardiac; Proteasome Endopeptidase Complex; Humans; Calcium; Animals; Arrhythmias, Cardiac; Induced Pluripotent Stem Cells; Stress, Physiological; Protein Transport; Rats; Amino Acids
PubMed: 38732146
DOI: 10.3390/ijms25094932 -
Biochemistry May 2024The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for...
The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DB1- and UL4-ssociated actors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-μM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.
Topics: Humans; DNA-Binding Proteins; Peptides; Fluorescence Polarization; Protein Binding; Ubiquitin-Protein Ligases
PubMed: 38729622
DOI: 10.1021/acs.biochem.4c00044 -
Bioorganic & Medicinal Chemistry Letters Jul 2024Targeted protein degradation is mediated by small molecules that induce or stabilize protein-protein interactions between targets and the ubiquitin-proteasome machinery....
Targeted protein degradation is mediated by small molecules that induce or stabilize protein-protein interactions between targets and the ubiquitin-proteasome machinery. Currently, there remains a need to expand the repertoire of viable E3 ligases available for hijacking. Notably, covalent chemistry has been employed to engage a handful of E3 ligases, including DCAF11. Here, we disclose a covalent PROTAC that enables DCAF11-dependent degradation, featuring a cyanoacrylamide warhead. Our findings underscore DCAF11 as an interesting candidate with a capacity to accommodate diverse electrophilic chemistries compatible with targeted protein degradation.
Topics: Humans; Acrylamides; Molecular Structure; Proteolysis; Drug Discovery; Ubiquitin-Activating Enzymes; Structure-Activity Relationship
PubMed: 38729317
DOI: 10.1016/j.bmcl.2024.129779 -
Cells Apr 2024In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae...
In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae may be a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. In this study, we investigated the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in the process of the transdifferentiation of auditory supporting cells (SCs) into hair cells (HCs). The expression of UCHL1 gradually decreased as HCs developed and was restricted to inner pillar cells and third-row Deiters' cells between P2 and P7, suggesting that UCHL1-expressing cells are similar to the cells with Lgr5-positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ-secretase inhibitor and Wnt agonist. Moreover, the inhibition of UCHL1 by LDN-57444 led to an increase in HC numbers. Mechanistically, LDN-57444 increased mTOR complex 1 activity and allowed SCs to transdifferentiate into HCs. The suppression of UCHL1 induces the transdifferentiation of auditory SCs and progenitors into HCs by regulating the mTOR pathway.
Topics: Animals; Cell Transdifferentiation; Hair Cells, Auditory; Indoles; Labyrinth Supporting Cells; Oximes; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin Thiolesterase; Rats
PubMed: 38727276
DOI: 10.3390/cells13090737 -
Clinical Transplantation and Research Mar 2024Following kidney transplantation, antibody-mediated rejection (AMR) occurs when the antibodies of the immune system attack the transplanted organ, leading to damage of... (Review)
Review
Following kidney transplantation, antibody-mediated rejection (AMR) occurs when the antibodies of the immune system attack the transplanted organ, leading to damage of the kidney tissue. human leukocyte antigen donor-specific antibodies (HLA-DSAs) play a key role in AMR. Current therapeutic approaches include intravenous immunoglobulin, anti-CD20 antibodies, and plasmapheresis. In cases resistant to treatment, proteasome inhibitors and C5 inhibitors may be employed. Nevertheless, a pressing need exists for new medications to improve transplant survival and reduce complications. In the context of AMR, interleukin (IL)-6 is instrumental in the development and maturation of B cells into plasma cells, which then produce HLA-DSAs targeting the allograft. IL-6 inhibitors are currently under investigation and show promise due to the essential role of IL-6 in the immune response; however, additional research is necessary.
PubMed: 38725179
DOI: 10.4285/ctr.23.0069 -
Nature Communications May 2024Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent...
Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.
Topics: Animals; Phosphorylation; NF-KappaB Inhibitor alpha; Mice; Transcription Factor RelA; Humans; Protein-Tyrosine Kinases; NF-kappa B; Cytoplasm; Proteolysis; Cell Nucleus; Virus Replication; HEK293 Cells; Signal Transduction; Mice, Inbred C57BL; Cytokines; Active Transport, Cell Nucleus; Protein Serine-Threonine Kinases
PubMed: 38724505
DOI: 10.1038/s41467-024-48288-z -
Cell Death Discovery May 2024Non-melanoma skin cancer (NMSC), encompassing basal and squamous cell carcinoma, is the most prevalent cancer in the United States. While surgical removal remains the...
Non-melanoma skin cancer (NMSC), encompassing basal and squamous cell carcinoma, is the most prevalent cancer in the United States. While surgical removal remains the conventional therapy with a 95% 5-year cure rate, there is a growing interest in exploring alternative treatment strategies. In this study, we investigated the role of Bortezomib (BTZ), a proteasome inhibitor, in NMSC. Using two NMSC cell lines (A431 and A388), we examined the effects of BTZ treatment. Our results demonstrated that 48 h of BTZ treatment led to downregulating Skp2 expression in both A431 and A388 cells while upregulating p53 expression, specifically in A388 cells. These alterations resulted in impaired cellular growth and caspase-dependent cell death. Silencing Skp2 in A388 cells with siRNA confirmed the upregulation of p53 as a direct target. Furthermore, BTZ treatment increased the Bax to Bcl-2 ratio, promoting mitochondrial permeability and the subsequent release of cytochrome C, thereby activating caspases. We also found that BTZ exerted its antitumor effects by generating reactive oxygen species (ROS), as blocking ROS production significantly reduced BTZ-induced apoptotic cell death. Interestingly, BTZ treatment induced autophagy, which is evident from the increased expression of microtubule-associated proteins nucleoporin p62 and LC-3A/B. In addition to cell lines, we assessed the impact of BTZ in an in vivo setting using Caenorhabditis elegans (C. elegans). Our findings demonstrated that BTZ induced germline apoptosis in worms even at low concentrations. Notably, this increased apoptosis was mediated through the activity of CEP-1, the worm's counterpart to mammalian p53. In summary, our study elucidated the molecular mechanism underlying BTZ-induced apoptosis in NMSC cell lines and C. elegans. By targeting the skp2/p53 axis, inducing mitochondrial permeability, generating ROS, and promoting autophagy, BTZ demonstrates promising anti-cancer activity in NMSC. These findings provide novel insights into potential therapeutic strategies for controlling the unregulated growth of NMSC.
PubMed: 38724504
DOI: 10.1038/s41420-024-01992-7