Proteasome-mediated Degradation Research Articles

The USP11/Nrf2 positive feedback loop promotes colorectal cancer progression by inhibiting mitochondrial apoptosis

AbstractAbnormal antioxidant capacity of cancer is closely related to tumor malignancy. Modulation of oxidative stress status is a novel anticancer therapeutic target. Nrf2 is a key regulator of various antioxidant enzymes, but the mechanism of its deubiquitination remains largely unclear. This study unveiled that Nrf2 received post-transcriptional regulation from a proteasome-associated deubiquitinating enzyme, USP11, in colorectal cancer (CRC). It was found that USP11 was overexpressed in CRC tissues acting as an oncogene by inhibiting mitochondrial apoptosis, and USP11 managed to maintain balance in the production and elimination of reactive oxygen species (ROS). Mechanistically, we identified a feedback loop between USP11 and Nrf2 maintaining the redox homeostasis. USP11 stabilized Nrf2 by deubiquitinating and protecting it from proteasome-mediated degradation. Interestingly, we also map that Nrf2 could bind to the antioxidant reaction element (ARE) in the USP11 promoter to promote its transcription. Hence, USP11/Nrf2 positive feedback loop inhibited mitochondrial apoptosis of CRC cells by activating Nrf2/ARE signaling pathway, thus promoting CRC progression.

Liquidambaric acid inhibits cholangiocarcinoma progression by disrupting the STAMBPL1/NRF2 positive feedback loop

BackgroundAbnormal antioxidant capacity in cancer cells is intimately linked to tumor aggressiveness. Modulating oxidative stress status and inhibiting ferroptosis represents a novel anticancer therapeutic strategy. STAM Binding Protein Like 1 (STAMBPL1), a deubiquitinase, is implicated in various malignancies, yet its function in inhibiting ferroptosis and therapeutic potential for cholangiocarcinoma (CCA) remains unexplored. Purpose: This study elucidates STAMBPL1's function in ferroptosis and evaluates liquidambaric acid (LDA) as its inhibitor for therapeutic applications. MethodsUsing bioinformatics, WB, IHC, the expression and prognostic value of STAMBPL1 in CCA tissue was detected. The carcinogenic capacity of STAMBPL1 and LDA were assessed through CCK-8, EdU, cloning, transwell, scratch, apoptosis, and cell cycle assays. Flow cytometry and fluorescence microscopy, as well as transmission electron microscopy (TEM), examines the effects of STAMBPL1 and LDA on intracellular reactive oxygen species (ROS) and changes in mitochondrial membrane potential. The tumorigenic ability of STAMBPL1 and LDA in vivo was evaluated through subcutaneous tumor model and lung metastasis model. The underlying mechanism of STAMBPL1 was explored using immunoprecipitation coupled with Mass spectrometry (IP/MS), Co-immunoprecipitation (Co-IP), GST pull-down, DNA pull-down, and Dual-luciferase reporter assays. Molecular docking simulations, SPR, DARTS and CETSA predict the putative binding site of LDA on STAMBPL1 protein. Rescue experiments further confirmed the above conclusions. ResultsThis study unveils the upregulation and oncogenic role of STAMBPL1 in CCA. Functionally, STAMBPL1 notably enhances CCA cell proliferation and metastasis while impeding ferroptosis. STAMBPL1 stabilizes NRF2, a pivotal regulator of antioxidant enzymes, through K63 deubiquitination. Elevated NRF2, stabilized by STAMBPL1 overexpression, triggers GPX4 activation and reactive oxygen species (ROS) elimination. Particularly, sites 251-436 of STAMBPL1 interact with sites 228-605 of NRF2, facilitating DUB activity and eliminating ubiquitin molecules attached to NRF2, thus protecting it from proteasome-mediated degradation. Moreover, NRF2, acting as a transcription factor, binds to the promoter region of STAMBPL1 and activates its transcription, thus forming STAMBPL1/NRF2 positive feedback loop and regulating redox homeostasis. Molecular docking and in vitro/in vivo experiments identified that LDA binds to and inhibits STAMBPL1, thereby disrupting the STAMBPL1/NRF2 positive feedback loop, consequently suppressing CCA progression. ConclusionThis study firstly reveals that STAMBPL1 promotes cholangiocarcinoma progression by upregulating NRF2, indicating that targeting the STAMBPL1/NRF2 axis is a novel therapeutic strategy. Additionally, our findings firstly suggest that LDA can bind to STAMBPL1, inhibiting NRF2 deubiquitination and offering significant therapeutic potential.

Neudesin, a secretory protein, attenuates activation of lipopolysaccharide-stimulated macrophages by suppressing the Jak/Stat1/iNOS pathway

AimsNeudesin, a heme-binding protein previously identified for its neurotrophic activity, has been implicated in various physiological and pathological processes, including immune regulation. However, its role in inflammatory macrophages remains unclear. Herein, we investigated the function of neudesin in the regulation of inflammatory macrophages. Main methodsIn vitro experiments were performed in bone marrow-derived macrophages (BMDMs). In vivo experiments were conducted on neudesin knockout mice with a murine endotoxic shock model. Key findingsWe observed that neudesin deficiency led to elevated expression of Nos2/iNOS and increased nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BMDMs. Further, we found that neudesin, via the ERK/MAPK signaling pathway, promotes the proteasome-mediated degradation of Stat1, resulting in suppression of NO production. Furthermore, neudesin-deficient mice exhibited higher mortality rates following LPS administration, accompanied by increased Nos2/iNOS expression and nitrated proteins in the heart, compared to that in wildtype mice. Treatment with an iNOS inhibitor drastically improved the survival rate of neudesin-deficient mice, highlighting the significance of neudesin-mediated iNOS signaling in modulating immune responses and preventing excessive inflammation. SignificanceOur findings suggest that neudesin acts as an anti-inflammatory cytokine, suppressing NO production in inflammatory macrophages, underscoring its potential as a therapeutic target for immune-related disorders.

Programmed cell death-related ABI1 is a critical mediator of abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a life-threatening condition characterized by localized dilation of the abdominal aorta, posing a significant risk of rupture and fatal hemorrhage. While surgical and endovascular repair techniques have advanced, the underlying mechanisms driving AAA development remain unclear, hindering the development of effective preventive and therapeutic strategies. Using bioinformatics analysis of publicly available data sets, the study identified a strong correlation between cell death (CD) score and different types of programmed cell death scores in AAA samples. WGCNA analysis revealed a module enriched in genes related to proteasome-mediated protein degradation, nuclear envelope, and endocytosis, significantly correlated with CD score. Further analysis identified ABI1 as a dominant feature gene, highlighting its potential role in AAA pathogenesis. In vitro validation using an Angiotensin II-induced AAA model in human aortic smooth muscle cells demonstrated that siRNA-mediated knockdown of ABI1 significantly reduced cell apoptosis, migration, and the expression of pro-apoptotic proteins, confirming ABI1's crucial role in promoting CD and AAA progression. The findings suggest that ABI1 may represent a promising therapeutic target for the prevention and treatment of AAA. Further research is warranted to fully understand the role of ABI1 in AAA and to develop targeted therapies based on this promising target.

SUMOylation of AL6 regulates seed dormancy and thermoinhibition in Arabidopsis.

DELAY OF GERMINATION1 (DOG1) is a critical regulator of seed dormancy and seed thermoinhibition. However, how DOG1 expression is regulated by post-translational modifications and how seeds transmit the high-temperature signal to DOG1 remain largely unknown. ALFIN1-like 6/7 (AL6/7) was previously found to repress DOG1 expression during seed imbibition. Here, we found that AL6/7 represses seed dormancy partly by downregulating DOG1 expression. AtSIZ1, a SUMO E3 ligase, interacts with AL6 and mediates its SUMOylation mainly at the lysine 181 residue. SIZ1-mediated SUMOylation of AL6 is required for repression of DOG1 transcription and seed dormancy. SUMOylation of AL6 is required for its association with the DOG1 locus and protects it from ubiquitination and subsequent 26S proteasome-mediated protein degradation. High temperatures decrease SUMOylation levels of AL6, resulting in downregulation of AL6 protein levels and an increase in DOG1 transcription, which consequently causes reduced seed germination. Taken together, these results demonstrate that reversible SUMOylation of AL6 fine-tunes DOG1 expression, which is required for precise establishment of seed dormancy and inhibition of seed germination under high-temperature conditions in Arabidopsis thaliana.

Exploring the Interactome of the Queuine Salvage Protein DUF2419 in Entamoeba histolytica.

Entamoeba histolytica causes amebiasis, a significant global health issue, with millions affected annually, especially in developing countries. EhDUF2419, an important protein involved in E. histolytica's queuine salvage pathway and its interaction network, remains unclear. To explore this, we transfected E. histolytica trophozoites with a plasmid encoding Myc-tagged EhDUF2419 and achieved successful overexpression. Through immunoprecipitation with the Myc antibody followed by mass spectrometry, we identified 335 proteins interacting with Myc-tagged EhDUF2419, including over 100 ribosomal proteins, along with translation initiation and elongation factors, and aminoacyl-tRNA synthetases. Ribosome purification revealed the presence of EhDUF2419 in ribosomal protein-enriched fractions. Treatment with queuosine (Q) significantly reduced the EhDUF2419 protein levels and decreased the Q-modified tRNA in Myc-tagged EhDUF2419 overexpressing trophozoites. This effect, which was Q-dependent, was not observed in strains carrying an empty vector control or overexpressing a truncated form of EhDUF2419 lacking catalytic activity. The reduction in the EhDUF2419 protein levels was regulated by proteasome-mediated degradation, as evidenced by the reduced degradation in the presence of MG132, a proteasome inhibitor. Our study uncovers the novel interaction of EhDUF2419 with ribosomal proteins and its regulation by the proteasome machinery, providing new insights into its role in E. histolytica and potential therapeutic strategies.

The transcription factor Dof3.6/OBP3 regulates iron homeostasis in Arabidopsis.

Iron is an essential element for plants. Iron uptake by plants is highly regulated, but the underlying mechanism is poorly understood. Using a truncated fragment of the iron deficiency-responsive bHLH100 gene promoter, we screened the Arabidopsis transcription factor yeast one-hybrid (Y1H) library and identified the DOF family protein, OBP3, as a crucial component of the iron deficiency-signaling pathway. OBP3 is a transcriptional repressor with a C-terminal activation domain. Its expression is induced by iron deficiency. The transgenic lines that overexpress OBP3 exhibited iron overload and premature leaf necrosis, while the obp3 mutant was less tolerant of iron deficiency. It was discovered that OBP3 directly targets the Ib subgroup of bHLH gene promoters. OBP3 interacts with the bHLH transcription factor ILR3 (IAA-LEUCINE RESISTANT3), and their interaction enhances the DNA-binding ability and transcriptional promoting activity of OBP3, resulting in the positive regulation of iron deficiency-response genes. In addition, the E3 Ligase BRUTUS facilitates 26S proteasome-mediated degradation of OBP3 protein to prevent excessive iron uptake in plants. In conclusion, our research emphasizes the vital role of OBP3 in regulating plant iron homeostasis.

Vitamin C Alleviates Heat-Stress-Induced Damages in Pig Thoracic Vertebral Chondrocytes via the Ubiquitin-Mediated Proteolysis Pathway.

Heat stress can impair organismal growth by inducing ubiquitination, proteasome-mediated degradation, and subsequent cellular damage. Vitamin C (VC) has been shown to potentially mitigate the detrimental effects of abiotic stresses on cells. Nevertheless, the impact of heat stress on growth plate chondrocytes remains unclear, and the underlying protective mechanisms of VC in these cells warrant further investigation. In this study, we focused on pig thoracic vertebral chondrocytes (PTVCs) that are crucial for promoting the body's longitudinal elongation and treated them with 41 °C heat stress for 24 h, under varying concentrations of VC. Our findings reveal that, while oxidative stress induced by heat triggers apoptosis and inhibits the ubiquitin-mediated proteolysis pathway, the addition of VC alleviates heat-stress-induced oxidative stress and apoptosis, mitigates cell cycle arrest, and promotes cellular viability. Furthermore, we demonstrate that VC enhances the ubiquitin-proteasome proteolysis pathway by promoting the expression of ubiquitin protein ligase E3A, which thereby stabilizes the ubiquitin-mediated degradation machinery, alleviates the apoptosis, and enhances cell proliferation. Our results suggest the involvement of the ubiquitin-mediated proteolysis pathway in the effects of VC on PTVCs under heat stress, and offer a potential strategy to make use of VC to ensure the skeletal growth of animals under high temperature pressures in summer or in tropical regions.

Single-cell multi-omics analysis of in vitro post-ovulatory aged oocytes revealed aging-dependent protein degradation

Once ovulated, the oocyte has to be fertilized in a short time window, or it will undergo post-ovulation aging (POA), whose underlying mechanisms are still not elucidated. Here, we optimized single-cell proteomics methods and performed single-cell transcriptomic, proteomic and phosphoproteomic analysis of fresh, POA, and melatonin-treated POA oocytes. POA oocytes showed down-regulation of most differentially expressed proteins, with little correlation with mRNA expression, and the protein changes can be rescued by melatonin treatment. MG132 treatment rescued the decreased fertilization and polyspermy rates, and up-regulated fragmentation and parthenogenesis rates of POA oocytes. MG132-treated oocytes displayed health status at proteome, phosphoproteome and fertilization ability similar to fresh oocytes, suggesting that protein stabilization might be the underlying mechanism for melatonin to rescue POA. The important roles of proteasome-mediated protein degradation during oocyte POA revealed by single-cell multi-omics analyses offer new perspectives for increasing oocyte quality during POA, and improving assisted reproduction technologies.

Enhanced cell stress response and protein degradation capacity underlie artemisinin resistance in Plasmodium falciparum.

Malaria remains a global health burden, killing over half a million people each year. Decreased therapeutic efficacy to artemisinin, the most efficacious antimalarial, has been detected in sub-Saharan Africa, a worrying fact given that over 90% of deaths occur on this continent. Mutations in Kelch13 are the most well-established molecular marker for artemisinin resistance, but these do not explain all artemisinin-resistant isolates. Understanding the biological underpinnings of drug resistance is key to curbing the emergence and spread of artemisinin resistance. Artemisinin-mediated non-specific alkylation leads to the accumulation of misfolded and damaged proteins and activation of the parasite unfolded protein response (UPR). In addition, the parasite proteasome is vital to artemisinin resistance, as we have previously shown that chemical inhibition of the proteasome or mutations in the β2 proteasome subunit increase parasite susceptibility to dihydroartemisinin (DHA), the active metabolite of artemisinins. Here, we investigate parasites with mutations at the Kelch13 and/or 19S and 20S proteasome subunits with regard to UPR regulation and proteasome activity in the context of artemisinin resistance. Our data show that perturbing parasite proteostasis kills parasites, early parasite UPR signaling dictates DHA survival outcomes, and DHA susceptibility correlates with impairment of proteasome-mediated protein degradation. Importantly, we show that functional proteasomes are required for artemisinin resistance in a Kelch13-independent manner, and compound-selective proteasome inhibition demonstrates why artemisinin-resistant Kelch13 mutants remain susceptible to the related antimalarial peroxide OZ439. These data provide further evidence for targeting the parasite proteasome and UPR to overcome existing artemisinin resistance.IMPORTANCEDecreased therapeutic efficacy represents a major barrier to malaria treatment control strategies. The malaria proteasome and accompanying unfolded protein response are crucial to artemisinin resistance, revealing novel antimalarial therapeutic strategies.

Development of 2-Aminoadenine-Based Proteolysis-Targeting Chimeras (PROTACs) as Novel Potent Degraders of Monopolar Spindle 1 and Aurora Kinases.

Monopolar spindle 1 (Mps1, also known as TTK) and Aurora kinase (AURK) A and B are critical regulators of mitosis and have been linked to the progression of various cancers. Here, we report the design, synthesis, and biological evaluation of a series of PROTACs (proteolysis-targeting chimeras) targeting TTK and AURKs. We synthesized various degrader molecules based on four different 2-aminoadenine-based ligands, recruiting either cereblon or VHL as the E3-ligase. Our research showed that the nature of the linker and modification of the ligand significantly influence the target specificity and degradation efficacy. Notably, compound 19, among the most potent degraders, demonstrated robust proteasome-mediated degradation of TTK with D max of 66.5% and DC50 value (6 h) of 17.7 nM as compared to its structurally akin inhibitor control, 23. The cytotoxicity of most of the synthesized chimeras against acute myeloid leukemia cell line MV4-11 was lower than that of the corresponding parent inhibitors. However, we could also identify degraders such as 15 and 26 that induce potent AURKA degradation and display comparable antiproliferative activities to their parent compound SF1. Compound 15 degrades AURKA with low DC50 value of 2.05 nM, which is 77-fold and 21-fold more selective toward AURKB and TTK and has an EC50 value of 39 nM against cancer MV4-11 cells. Overall, the observations we made with the degrader molecules we developed can further aid in the design and development of optimized TTK or AURK degraders for cancer therapy.

Abstract IA01: Dumpster-diving into the cancer degradome

Abstract Recently, we uncovered a pivotal role for altered proteasome complex composition in shaping tumor immunogenicity in non-small cell lung cancer (NSCLC). Our research demonstrated that the upregulation of a regulatory cap of the proteasome, PSME4, leads to a reduction in antigenic diversity through alterations in proteasomal cleavage patterns, consequently impeding T-cell-mediated anti-tumor immunity. These findings underscore the importance of probing the tumor degradome and its implications for tumor-immune interactions. Yet, numerous questions remain unanswered: Do specific substrates exhibit preferential degradation in PSME4-capped proteasomes? What constitutes their recognition signal? And fundamentally, how do alterations in the degradation landscape influence tumor inflammation? In my talk, I will delve into our latest advancements in addressing these questions, offering insights into the intricate interplay between proteasome-mediated degradation, tumor inflammation, and STAT signaling. Citation Format: Yifat Merbl. Dumpster-diving into the cancer degradome [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr IA01.

RhoBTB3 Functions as a Novel Regulator of Autophagy by Suppressing AMBRA1 Stability.

Autophagy is essential for cell survival and cellular homeostasis under various stress conditions. Therefore, autophagy dysfunction is associated with the pathogenesis of various human diseases. We explored the regulatory role of RhoBTB3 in autophagy and its interaction with activating molecules in AMBRA1. RhoBTB3 deficiency was found to induce autophagy, while its overexpression inhibited autophagy induction. Through immunoprecipitation and mass spectrometry, AMBRA1 was identified as a substrate of RhoBTB3. The study revealed that RhoBTB3 regulates AMBRA1 stability by influencing its protein levels without affecting its mRNA levels. RhoBTB3 induced the ubiquitination of AMBRA1, leading to proteasome-mediated degradation, with the ubiquitination occurring at K45 on AMBRA1 through a K27-linked ubiquitin chain. The knockdown of AMBRA1 blocked RhoBTB3 knockdown-induced autophagy, indicating the dependency of autophagy on AMBRA1. Thus, RhoBTB3 negatively regulates autophagy by mediating AMBRA1 ubiquitination and degradation, suggesting RhoBTB3 as a potential therapeutic target for autophagy-related diseases.

A Structural Investigation of the Interaction between a GC-376-Based Peptidomimetic PROTAC and Its Precursor with the Viral Main Protease of Coxsackievirus B3

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CLPro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3CPro protease of enteroviruses, specifically characterizing its interaction with CVB3 3CPro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3CPro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3CPro and SARS-CoV-2 3CLPro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3CPro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3CPro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3CPro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent.

The potato RNA metabolism machinery is targeted by the cyst nematode effector RHA1B for successful parasitism.

The potato (Solanum tuberosum) cyst nematode Globodera pallida induces a multinucleate feeding site (syncytium) in potato roots as its sole source of nutrition. Here, we demonstrate that the G. pallida effector RING-H2 finger A1b (RHA1B), which is a functional ubiquitin ligase, interferes with the carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT) deadenylase-based RNA metabolism machinery that regulates syncytium development in G. pallida-infected potato. Specifically, RHA1B targets the CCR4-associated factor 1 (CAF1) and StNOT10 subunits of the CCR4-NOT complex for proteasome-mediated degradation, leading to upregulation of the cyclin gene StCycA2 involved in syncytium formation. The StCAF1 subunit of CCR4-NOT recruits the RNA binding protein StPUM5 to deadenylate StCycA2 mRNA, resulting in shortened poly-A tails of StCycA2 mRNA and subsequently reduced transcript levels. Knockdown of either subunit (StCAF1 or StNOT10) of the CCR4-NOT complex or StPUM5 in transgenic potato plants resulted in enlarged syncytia and enhanced susceptibility to G. pallida infection, which resembles the phenotypes of StCycA2 overexpression transgenic potato plants. Genetic analyses indicate that transgenic potato plants overexpressing RHA1B exhibit similar phenotypes as transgenic potato plants with knockdown of StNOT10, StCAF1, or StPUM5. Thus, our data suggest that G. pallida utilizes the RHA1B effector to manipulate RNA metabolism in host plants, thereby promoting syncytium development for parasitic success.

Stub1 promotes degradation of the activated Diaph3: A negative feedback regulatory mechanism of the actin nucleator

The formin protein Diaph3 is an actin nucleator that regulates numerous cytoskeleton-dependent cellular processes through the activation of actin polymerization. Expression and activity of Diaph3 is tightly regulated: lack of Diaph3 results in developmental defects and embryonic lethality in mice, while overexpression of Diaph3 causes auditory neuropathy. It is known that Diaph3 homophilic interactions include the intramolecular interaction of its DID-DAD domains and the intermolecular interactions of DD-DD domains or FH2-FH2 domains. However, the physiological significance of these interactions in Diaph3 protein stability and activity is not fully understood. In this study, we show that FH2-FH2 interaction promotes Diaph3 activity, while DID-DAD and DD-DD interactions inhibit Diaph3 activity through distinct mechanisms. DID-DAD interaction is responsible for the autoinhibition of Diaph3 protein, which is disrupted by binding of Rho GTPases. Interestingly, we find that DID-DAD interaction stabilizes the expression of each DID or DAD domain against proteasomal-mediated degradation. Disruption of DID-DAD interaction by RhoA binding or M1041A mutation causes increased Diaph3 activity and accelerated degradation of the activated Diaph3 protein. Further, the activated Diaph3 is ubiquitinated at K1142/1143/1144 lysine residues by the E3 ligase Stub1. Expression of Stub1 is causally related to the stability and activity of Diaph3. Knockdown of Stub1 in mouse cochlea results in hair cell stereocilia defects, neuronal degeneration and hearing loss, resembling the phenotypes of mice overexpressing Diaph3. Thus, our study reports a novel regulatory mechanism of Diaph3 protein expression and activity whereby the active but not inactive Diaph3 is readily degraded to prevent excessive actin polymerization.

Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation

A dynamic proteome is required for cellular adaption to changing environments including levels of O2, and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O2. Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O2-dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O2 as an environmental signal.

Development of a monoclonal antibody to study MARCH6, an E3 ligase that regulates proteins that control lipid homeostasis

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

The protective role of RACK1 in hepatic ischemia‒reperfusion injury-induced ferroptosis.

Although ferroptosis plays a crucial role in hepatic ischemia‒reperfusion injury (IRI), the molecular mechanisms underlying this process remain unclear. We aimed to explore the potential involvement of the receptor for activated C kinase 1 (RACK1) in hepatic IRI-triggered ferroptosis. Using hepatocyte-specific RACK1 knockout mice and alpha mouse liver 12 (AML12) cells, we conducted a series of in vivo and in vitro experiments. We found that RACK1 has a protective effect on hepatic IRI-induced ferroptosis. Specifically, RACK1 was found to interact with AMPKα through its 1-93 amino acid (aa) region, which facilitates the phosphorylation of AMPKα at threonine 172 (Thr172), ultimately exerting an antiferroptotic effect. Furthermore, the long noncoding RNA (lncRNA) ZNFX1 Antisense 1 (ZFAS1) directly binds to aa 181-317 of RACK1. ZFAS1 has a dual impact on RACK1 by promoting its ubiquitin‒proteasome-mediated degradation and inhibiting its expression at the transcriptional level, which indirectly exacerbates hepatic IRI-induced ferroptosis. These findings underscore the protective role of RACK1 in hepatic IRI-induced ferroptosis and showcase its potential as a prophylactic target for hepatic IRI mitigation.

Acidity suppresses CD8 + T-cell function by perturbing IL-2, mTORC1, and c-Myc signaling

CD8 + T cells have critical roles in tumor control, but a range of factors in their microenvironment such as low pH can suppress their function. Here, we demonstrate that acidity restricts T-cell expansion mainly through impairing IL-2 responsiveness, lowers cytokine secretion upon re-activation, and reduces the cytolytic capacity of CD8 + T cells expressing low-affinity TCR. We further find decreased mTORC1 signaling activity and c-Myc levels at low pH. Mechanistically, nuclear/cytoplasmic acidification is linked to mTORC1 suppression in a Rheb-, Akt/TSC2/PRAS40-, GATOR1- and Lkb1/AMPK-independent manner, while c-Myc levels drop due to both decreased transcription and higher levels of proteasome-mediated degradation. In addition, lower intracellular levels of glutamine, glutamate, and aspartate, as well as elevated proline levels are observed with no apparent impact on mTORC1 signaling or c-Myc levels. Overall, we suggest that, due to the broad impact of acidity on CD8 + T cells, multiple interventions will be required to restore T-cell function unless intracellular pH is effectively controlled.