Stress Granule Core Protein-Derived Peptides Inhibit Assembly of Stress Granules and Improve Sorafenib Sensitivity in Cancer Cells.

Upon a variety of environmental stresses, eukaryotic cells usually recruit translational stalled mRNAs and RNA-binding proteins to form cytoplasmic condensates known as stress granules (SGs), which minimize stress-induced damage and promote stress adaptation and cell survival. SGs are hijacked by cancer cells to promote cell survival and are consequently involved in the development of anticancer drug resistance. However, the design and application of chemical compounds targeting SGs to improve anticancer drug efficacy have rarely been studied. Here, we developed two types of SG inhibitory peptides (SIPs) derived from SG core proteins Caprin1 and USP10 and fused with cell-penetrating peptides to generate TAT-SIP-C1/2 and SIP-U1-Antp, respectively. We obtained 11 SG-inducing anticancer compounds from cell-based screens and explored the potential application of SIPs in overcoming resistance to the SG-inducing anticancer drug sorafenib. We found that SIPs increased the sensitivity of HeLa cells to sorafenib via the disruption of SGs. Therefore, anticancer drugs which are competent to induce SGs could be combined with SIPs to sensitize cancer cells, which might provide a novel therapeutic strategy to alleviate anticancer drug resistance.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, 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 P. falciparum 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 Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Metrnl inhibits choroidal neovascularization by attenuating the choroidal inflammation via inactivating the UCHL-1/NF-κB signaling pathway.

Objective: Choroidal neovascularization (CNV) represents the predominant form of advanced wet Age-related Macular Degeneration (wAMD). Macrophages play a pivotal role in the pathological progression of CNV. Meteorin-like (Metrnl), a novel cytokine known for its anti-inflammatory properties in macrophages, is the focus of our investigation into its mechanism of action and its potential to impede CNV progression. Methods: Cell viability was evaluated through CCK-8 and EdU assays following Metrnl treatment. Expression levels of inflammatory cytokines and proteins were assessed using quantitative reverse-transcription polymerase chain reaction(qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot techniques. Protein-protein interactions were identified through protein mass spectrometry and co-immunoprecipitation (Co-IP). Additionally, in vivo and in vitro neovascularization models were employed to evaluate angiogenesis. Results: Our results revealed downregulated Metrnl levels in the choroid-sclera complex of CNV mice, the aqueous humor of wAMD patients, and activated macrophages. Metrnl overexpression demonstrated a reduction in pro-inflammatory cytokine production, influenced endothelial cell function, and suppressed angiogenesis in choroid explants and CNV models. Through protein mass spectrometry and Co-IP, we confirmed Metrnl binds to UCHL-1 to modulate the NF-κB signaling pathway. This interaction inhibited the transcription and expression of pro-inflammatory cytokines, ultimately suppressing angiogenesis. Conclusion: In summary, our findings indicate that Metrnl down-regulates macrophage pro-inflammatory cytokine secretion via the UCHL-1/NF-κB signaling pathway. This mechanism alleviates the inflammatory microenvironment and effectively inhibits choroidal neovascularization.

The Suppression of Ubiquitin C-Terminal Hydrolase L1 Promotes the Transdifferentiation of Auditory Supporting Cells into Hair Cells by Regulating the mTOR Pathway.

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.

Deficiency of UCHL1 results in insufficient decidualization accompanied by impaired dNK modulation and eventually miscarriage.

BACKGROUND: Miscarriage is a frustrating complication of pregnancy that is common among women of reproductive age. Insufficient decidualization which not only impairs embryo implantation but disturbs fetomaternal immune-tolerance, has been widely regarded as a major cause of miscarriage; however, the underlying mechanisms resulting in decidual impairment are largely unknown. METHODS: With informed consent, decidual tissue from patients with spontaneous abortion or normal pregnant women was collected to detect the expression profile of UCHL1. Human endometrial stromal cells (HESCs) were used to explore the roles of UCHL1 in decidualization and dNK modulation, as well as the mechanisms involved. C57/BL6 female mice (7-10 weeks old) were used to construct pregnancy model or artificially induced decidualization model to evaluate the effect of UCHL1 on mice decidualization and pregnancy outcome. RESULTS: The Ubiquitin C-terminal hydrolase L1 (UCHL1), as a deubiquitinating enzyme, was significantly downregulated in decidua from patients with miscarriage, along with impaired decidualization and decreased dNKs. Blockage of UCHL1 led to insufficient decidualization and resultant decreased expression of cytokines CXCL12, IL-15, TGF-ß which were critical for generation of decidual NK cells (dNKs), whereas UCHL1 overexpression enhanced decidualization accompanied by increase in dNKs. Mechanistically, the promotion of UCHL1 on decidualization was dependent on its deubiquitinating activity, and intervention of UCHL1 inhibited the activation of JAK2/STAT3 signaling pathway, resulting in aberrant decidualization and decreased production of cytokines associated with dNKs modulation. Furthermore, we found that inhibition of UCHL1 also disrupted the decidualization in mice and eventually caused adverse pregnancy outcome. CONCLUSIONS: UCHL1 plays significant roles in decidualization and dNKs modulation during pregnancy in both humans and mice. Its deficiency indicates a poor pregnancy outcome due to defective decidualization, making UCHL1 a potential target for the diagnosis and treatment of miscarriage.

USP22 as a key regulator of glycolysis pathway in osteosarcoma: insights from bioinformatics and experimental approaches.

Background: Osteosarcoma is the most common primary malignant bone tumor, but its pathogenesis remains unclear. Ubiquitin-specific processing peptidase 22 (USP22) is reported to be highly expressed and associated with tumor malignancy and prognosis in cancers. However, the role and mechanism of USP22 in osteosarcoma is not fully understood. This study aims to investigate the function and potential mechanism of USP22 in osteosarcoma using bioinformatics analysis combined with experimental validation. Methods: We first integrated transcriptomic datasets and clinical information of osteosarcoma from GEO and TCGA databases to assess the expression and prognostic value of USP22 in osteosarcoma. Then, differential expression analysis and weighted gene co-expression network analysis (WGCNA) were conducted to identify USP22-related co-expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore the biological functions and signaling pathways of USP22 co-expressed genes. To validate the accuracy of bioinformatics analyses, we downregulated USP22 expression in osteosarcoma cell line Sao-2 using siRNA and assessed its effect on cell proliferation, migration, invasion, apoptosis, and regulation of key signaling pathways. Results: We found that USP22 was highly expressed in osteosarcoma tissues and correlated with poor prognosis in osteosarcoma patients. USP22 also showed potential as a diagnostic marker for osteosarcoma. In addition, 344 USP22-related co-expressed genes were identified, mainly involved in signaling pathways such as glycolysis, oxidative phosphorylation, spliceosome, thermogenesis, and cell cycle. The in vitro experiments confirmed the accuracy and reliability of bioinformatics analyses. We found that downregulation of USP22 could inhibit Sao-2 cell proliferation, migration, invasion, and induce apoptosis. Furthermore, downregulation of USP22 significantly reduced aerobic glycolysis levels in Sao-2 cells and inhibited the expression of key enzymes and transporters in aerobic glycolysis pathways such as HK2, PKM2, and GLUT1. Conclusions: USP22 plays a critical role in the occurrence, development, and prognosis of osteosarcoma. USP22 could influence Sao-2 cell proliferation, apoptosis, migration, and invasion by regulating the glycolysis pathway, thereby promoting osteosarcoma progression. Therefore, USP22 may be a potential therapeutic target for the treatment of osteosarcoma.

The Emerging Role of Ubiquitin-Specific Protease 36 (USP36) in Cancer and Beyond.

The balance between ubiquitination and deubiquitination is instrumental in the regulation of protein stability and maintenance of cellular homeostasis. The deubiquitinating enzyme, ubiquitin-specific protease 36 (USP36), a member of the USP family, plays a crucial role in this dynamic equilibrium by hydrolyzing and removing ubiquitin chains from target proteins and facilitating their proteasome-dependent degradation. The multifaceted functions of USP36 have been implicated in various disease processes, including cancer, infections, and inflammation, via the modulation of numerous cellular events, including gene transcription regulation, cell cycle regulation, immune responses, signal transduction, tumor growth, and inflammatory processes. The objective of this review is to provide a comprehensive summary of the current state of research on the roles of USP36 in different pathological conditions. By synthesizing the findings from previous studies, we have aimed to increase our understanding of the mechanisms underlying these diseases and identify potential therapeutic targets for their treatment.

Nerve Bundle Density and Expression of NGF and IL-1ß Are Intra-Individually Heterogenous in Subtypes of Endometriosis.

Endometriosis is a gynecological disorder associated with local inflammation and neuroproliferation. Increased nerve bundle density has been attributed to increased expression of nerve growth factor (NGF) and interleukin-1ß (IL-1ß). Immunohistochemical analysis was carried out on 12 patients presenting with all three anatomic subtypes of endometriosis (deep, superficial peritoneal, endometrioma) at surgery, with at least two surgically excised subtypes available for analysis. Immunolocalization for nerve bundle density around endometriosis using protein gene product 9.5 (PGP9.5), as well as NGF and IL-1ß histoscores in endometriosis epithelium/stroma, was performed to evaluate differences in scores between lesions and anatomic subtypes per patient. Intra-individual heterogeneity in scores across lesions was assessed using the coefficient of variation (CV). The degree of score variability between subtypes was evaluated using the percentage difference between mean scores from one subtype to another subtype for each marker. PGP9.5 nerve bundle density was heterogenous across multiple subtypes of endometriosis, ranging from 50.0% to 173.2%, where most patients (8/12) showed CV ≥ 100%. The percentage difference in scores showed that PGP9.5 nerve bundle density and NGF and IL-1ß expression were heterogenous between anatomic subtypes within the same patient. Based on these observations of intra-individual heterogeneity, we conclude that markers of neuroproliferation in endometriosis should be stratified by anatomic subtype in future studies of clinical correlation.

USP18 Curbs the Progression of Metabolic Hypertension by Suppressing JAK/STAT Pathway.

Hypertension is a pathological state of the metabolic syndrome that increases the risk of cardiovascular disease. Managing hypertension is challenging, and we aimed to identify the pathogenic factors and discern therapeutic targets for metabolic hypertension (MHR). An MHR rat model was established with the combined treatment of a high-sugar, high-fat diet and ethanol. Histopathological observations were performed using hematoxylin-eosin and Sirius Red staining. Transcriptome sequencing was performed to screen differentially expressed genes. The role of ubiquitin-specific protease 18 (USP18) in the proliferation, apoptosis, and oxidative stress of HUVECs was explored using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays. Moreover, USP18 downstream signaling pathways in MHR were screened, and the effects of USP18 on these signaling pathways were investigated by western blotting. In the MHR model, total cholesterol and low-density lipoprotein levels increased, while high-density lipoprotein levels decreased. Moreover, high vessel thickness and percentage of collagen were noted along with increased malondialdehyde, decreased superoxide dismutase and catalase levels. The staining results showed that the MHR model exhibited an irregular aortic intima and disordered smooth muscle cells. There were 78 differentially expressed genes in the MHR model, and seven hub genes, including USP18, were identified. USP18 overexpression facilitated proliferation and reduced apoptosis and oxidative stress in HUVECs treated with Ang in vitro. In addition, the JAK/STAT pathway was identified as a USP18 downstream signaling pathway, and USP18 overexpression inhibited the expression of JAK/STAT pathway-related proteins. Conclusively, USP18 restrained MHR progression by promoting cell proliferation, reversing apoptosis and oxidative stress, and suppressing the JAK/STAT pathway.

UCHL5 promotes hepatocellular carcinoma progression by promoting glycolysis through activating Wnt/ß-catenin pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is highly malignant with a dismal prognosis, although the available therapies are insufficient. No efficient ubiquitinase has been identified as a therapeutic target for HCC despite the complicating role that of proteins ubiquitination plays in the malignant development of HCC. METHODS: The expression of ubiquitin carboxyl terminal hydrolase L5 (UCHL5) in HCC tumor tissue and adjacent normal tissue was determined using the cancer genome atlas (TCGA) database and was validated using real-time quantitative polymerase chain reaction (RT-qRCR), Western blot and immunohistochemistry (IHC), and the relation of UCHL5 with patient clinical prognosis was explored. The expression of UCHL5 was knocked down and validated, and the effect of UCHL5 on the biological course of HCC was explored using cellular assays. To clarify the molecular mechanism of action of UCHL5 affecting HCC, expression studies of Adenosine triphosphate adenosine triphosphate (ATP), extracellular acidification (ECAR), and glycolysis-related enzymes were performed. The effects of UCHL5 on ß-catenin ubiquitination and Wnt signaling pathways were explored in depth and validated using cellular functionalities. Validation was also performed in vivo. RESULTS: In the course of this investigation, we discovered that UCHL5 was strongly expressed in HCC at both cellular and tissue levels. The prognosis of patients with high UCHL5 expression is considerably worse than that of those with low UCHL5 expression. UCHL5 has been shown to increase the degree of glycolysis in HCC cells with the impact of stimulating the proliferation and metastasis of HCC cells in both in vivo and in vitro. UCHL5 downregulates its degree of ubiquitination by binding to ß-catenin, which activates the Wnt/ß-catenin pathway and accelerates HCC cell glycolysis. Thereby promoting the growth of the HCC. CONCLUSIONS: In summary, we have demonstrated for the first time that UCHL5 is a target of HCC and promotes the progression of hepatocellular carcinoma by promoting glycolysis through the activation of the Wnt/ß-catenin pathway. UCHL5 may thus serve as a novel prognostic marker and therapeutic target for the treatment of HCC.

Usp14 deficiency removes α-synuclein by regulating S100A8/A9 in Parkinson's disease.

Ubiquitin-proteasome system dysfunction triggers α-synuclein aggregation, a hallmark of neurodegenerative diseases, such as Parkinson's disease (PD). However, the crosstalk between deubiquitinating enzyme (DUBs) and α-synuclein pathology remains unclear. In this study, we observed a decrease in the level of ubiquitin-specific protease 14 (USP14), a DUB, in the cerebrospinal fluid (CSF) of PD patients, particularly females. Moreover, CSF USP14 exhibited a dual correlation with α-synuclein in male and female PD patients. To investigate the impact of USP14 deficiency, we crossed USP14 heterozygous mouse (USP14+/-) with transgenic A53T PD mouse (A53T-Tg) or injected adeno-associated virus (AAV) carrying human α-synuclein (AAV-hα-Syn) in USP14+/- mice. We found that Usp14 deficiency improved the behavioral abnormities and pathological α-synuclein deposition in female A53T-Tg or AAV-hα-Syn mice. Additionally, Usp14 inactivation attenuates the pro-inflammatory response in female AAV-hα-Syn mice, whereas Usp14 inactivation demonstrated opposite effects in male AAV-hα-Syn mice. Mechanistically, the heterodimeric protein S100A8/A9 may be the downstream target of Usp14 deficiency in female mouse models of α-synucleinopathies. Furthermore, upregulated S100A8/A9 was responsible for α-synuclein degradation by autophagy and the suppression of the pro-inflammatory response in microglia after Usp14 knockdown. Consequently, our study suggests that USP14 could serve as a novel therapeutic target in PD.

Ruthenium red alleviates post-resuscitation myocardial dysfunction by upregulating mitophagy through inhibition of USP33 in a cardiac arrest rat model.

Cardiac arrest (CA) remains a leading cause of death, with suboptimal survival rates despite efforts involving cardiopulmonary resuscitation and advanced life-support technology. Post-resuscitation myocardial dysfunction (PRMD) is an important determinant of patient outcomes. Myocardial ischemia/reperfusion injury underlies this dysfunction. Previous reports have shown that ruthenium red (RR) has a protective effect against cardiac ischemia-reperfusion injury; however, its precise mechanism of action in PRMD remains unclear. This study investigated the effects of RR on PRMD and analyzed its underlying mechanisms. Ventricular fibrillation was induced in rats, which were then subjected to cardiopulmonary resuscitation to establish an experimental CA model. At the onset of return of spontaneous circulation, RR (2.5 mg/kg) was administered intraperitoneally. Our study showed that RR improved myocardial function and reduced the production of oxidative stress markers such as malondialdehyde (MDA), glutathione peroxidase (GSSG), and reactive oxygen species (ROS) production. RR also helped maintain mitochondrial structure and increased ATP and GTP levels. Additionally, RR effectively attenuated myocardial apoptosis. Furthermore, we observed downregulation of proteins closely related to mitophagy, including ubiquitin-specific protease 33 (USP33) and P62, whereas LC3B (microtubule-associated protein light chain 3B) was upregulated. The upregulation of mitophagy may play a critical role in reducing myocardial injury. These results demonstrate that RR may attenuate PRMD by promoting mitophagy through the inhibition of USP33. These effects are likely mediated through diverse mechanisms, including antioxidant activity, apoptosis suppression, and preservation of mitochondrial integrity and energy metabolism. Consequently, RR has emerged as a promising therapeutic approach for addressing post-resuscitation myocardial dysfunction.

Proteostatic reactivation of the developmental transcription factor TBX3 drives BRAF/MAPK-mediated tumorigenesis.

MAPK pathway-driven tumorigenesis, often induced by BRAFV600E, relies on epithelial dedifferentiation. However, how lineage differentiation events are reprogrammed remains unexplored. Here, we demonstrate that proteostatic reactivation of developmental factor, TBX3, accounts for BRAF/MAPK-mediated dedifferentiation and tumorigenesis. During embryonic development, BRAF/MAPK upregulates USP15 to stabilize TBX3, which orchestrates organogenesis by restraining differentiation. The USP15-TBX3 axis is reactivated during tumorigenesis, and Usp15 knockout prohibits BRAFV600E-driven tumor development in a Tbx3-dependent manner. Deleting Tbx3 or Usp15 leads to tumor redifferentiation, which parallels their overdifferentiation tendency during development, exemplified by disrupted thyroid folliculogenesis and elevated differentiation factors such as Tpo, Nis, Tg. The clinical relevance is highlighted in that both USP15 and TBX3 highly correlates with BRAFV600E signature and poor tumor prognosis. Thus, USP15 stabilized TBX3 represents a critical proteostatic mechanism downstream of BRAF/MAPK-directed developmental homeostasis and pathological transformation, supporting that tumorigenesis largely relies on epithelial dedifferentiation achieved via embryonic regulatory program reinitiation.

BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system.

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

Hypoxic adipose stem cell-derived exosomes carrying high-abundant USP22 facilitate cutaneous wound healing through stabilizing HIF-1α and upregulating lncRNA H19.

Hypoxic preconditioning has been recognized as a promotive factor for accelerating cutaneous wound healing. Our previous study uncovered that exosomal lncRNA H19, derived from adipose-derived stem cells (ADSCs), plays a crucial role in orchestrating cutaneous wound healing. Herein, we aimed to explore whether there is a connection between hypoxia and ADSC-derived exosomes (ADSCs-exos) in cutaneous wound healing. Exosomes extracted from ADSCs under normoxic and hypoxic conditions were identified using transmission electron microscope (TEM) and particle size analysis. The effects of ADSCs-exos on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, EdU, wound healing, and tube formation assays. Expression patterns of H19, HIF-1α, and USP22 were measured. Co-immunoprecipitation, chromatin immunoprecipitation, ubiquitination, and luciferase reporter assays were conducted to confirm the USP22/HIF-1α/H19 axis, which was further validated in a mice model of skin wound. Exosomes extracted from hypoxia-treated ADSCs (termed as H-ADSCs-exos) significantly increased cell proliferation, migration, and angiogenesis in H2O2-exposed HUVECs, and promoted cutaneous wound healing in vivo. Moreover, H-ADSCs and H-ADSCs-exos, which exhibited higher levels of H19, were found to be transcriptionally activated by HIF-1α. Mechanically, H-ADSCs carrying USP22 accounted for deubiquitinating and stabilizing HIF-1α. Additionally, H-ADSCs-exos improved cell proliferation, migration, and angiogenesis in H2O2-triggered HUVECs by activating USP22/HIF-1α axis and promoting H19 expression, which may provide a new clue for the clinical treatment of cutaneous wound healing.

Performance of glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) biomarkers in predicting CT scan results and neurological outcomes in children with traumatic brain injury (BRAINI-2 paediatric study): protocol of a European prospective multicentre study.

INTRODUCTION: In light of the burden of traumatic brain injury (TBI) in children and the excessive number of unnecessary CT scans still being performed, new strategies are needed to limit their use while minimising the risk of delayed diagnosis of intracranial lesions (ICLs). Identifying children at higher risk of poor outcomes would enable them to be better monitored. The use of the blood-based brain biomarkers glial fibrillar acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) could help clinicians in this decision. The overall aim of this study is to provide new knowledge regarding GFAP and UCH-L1 in order to improve TBI management in the paediatric population. METHODS AND ANALYSIS: We will conduct a European, prospective, multicentre study, the BRAINI-2 paediatric study, in 20 centres in France, Spain and Switzerland with an inclusion period of 30 months for a total of 2880 children and adolescents included. To assess the performance of GFAP and UCH-L1 used separately and in combination to predict ICLs on CT scans (primary objective), 630 children less than 18 years of age with mild TBI, defined by a Glasgow Coma Scale score of 13-15 and with a CT scan will be recruited. To evaluate the potential of GFAP and UCH-L1 in predicting the prognosis after TBI (secondary objective), a further 1720 children with mild TBI but no CT scan as well as 130 children with moderate or severe TBI will be recruited. Finally, to establish age-specific reference values for GFAP and UCH-L1 (secondary objective), we will include 400 children and adolescents with no history of TBI. ETHICS AND DISSEMINATION: This study has received ethics approval in all participating countries. Results from our study will be disseminated in international peer-reviewed journals. All procedures were developed in order to assure data protection and confidentiality. TRIAL REGISTRATION NUMBER: NCT05413499.

GZ17-6.02 kills PDX isolates of uveal melanoma.

GZ17-6.02 has undergone phase I evaluation in patients with solid tumors (NCT03775525). The RP2D is 375 mg PO BID, with an uveal melanoma patient exhibiting a 15% reduction in tumor mass for 5 months at this dose. Studies in this manuscript have defined the biology of GZ17-6.02 in PDX isolates of uveal melanoma cells. GZ17-6.02 killed uveal melanoma cells through multiple convergent signals including enhanced ATM-AMPK-mTORC1 activity, inactivation of YAP/TAZ and inactivation of eIF2α. GZ17-6.02 significantly enhanced the expression of BAP1, predictive to reduce metastasis, and reduced the levels of ERBB family RTKs, predicted to reduce growth. GZ17-6.02 interacted with doxorubicin or ERBB family inhibitors to significantly enhance tumor cell killing which was associated with greater levels of autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5 or eIF2α were more protective than knock down of ATM, AMPKα, CD95 or FADD, however, over-expression of FLIP-s provided greater protection compared to knock down of CD95 or FADD. Expression of activated forms of mTOR and STAT3 significantly reduced tumor cell killing. GZ17-6.02 reduced the expression of PD-L1 in uveal melanoma cells to a similar extent as observed in cutaneous melanoma cells whereas it was less effective at enhancing the levels of MHCA. The components of GZ17-6.02 were detected in tumors using a syngeneic tumor model. Our data support future testing GZ17-6.02 in uveal melanoma as a single agent, in combination with ERBB family inhibitors, in combination with cytotoxic drugs, or with an anti-PD1 immunotherapy.

Regulation of ubiquitination and antiviral activity of Cactin by deubiquitinase Usp14 in Drosophila.

Cactin, a highly conserved protein, plays a crucial role in various physiological processes in eukaryotes, including innate immunity. Recently, the function of Cactin in the innate immunity of Drosophila has been explored, revealing that Cactin regulates a non-canonical signaling pathway associated with the Toll and Imd pathways via the Cactin-Deaf1 axis. In addition, Cactin exhibits specific antiviral activity against the Drosophila C virus (DCV) in Drosophila, with an unknown mechanism. During DCV infection, it has been confirmed that the protein level and antiviral activity of Cactin are regulated by ubiquitination. However, the precise ubiquitination and deubiquitination mechanisms of Cactin in Drosophila remain unexplored. In this study, we identified ubiquitin-specific protease 14 (Usp14) as a major deubiquitinase for Cactin through comprehensive deubiquitinase screening. Our results demonstrate that Usp14 interacts with the C_Cactus domain of Cactin via its USP domain. Usp14 efficiently removes K48- and K63-linked polyubiquitin chains from Cactin, thereby preventing its degradation through the ubiquitin-proteasome pathway. Usp14 significantly inhibits DCV replication in Drosophila cells by stabilizing Cactin. Moreover, Usp14-deficient fruit flies exhibit increased susceptibility to DCV infection compared to wild-type flies. Collectively, our findings reveal the regulation of ubiquitination and antiviral activity of Cactin by the deubiquitinase Usp14, providing valuable insights into the modulation of Cactin-mediated antiviral activity in Drosophila.IMPORTANCEViral infections pose a severe threat to human health, marked by high pathogenicity and mortality rates. Innate antiviral pathways, such as Toll, Imd, and JAK-STAT, are generally conserved across insects and mammals. Recently, the multi-functionality of Cactin in innate immunity has been identified in Drosophila. In addition to regulating a non-canonical signaling pathway through the Cactin-Deaf1 axis, Cactin exhibits specialized antiviral activity against the Drosophila C virus (DCV) with an unknown mechanism. A previous study emphasized the significance of the Cactin level, regulated by the ubiquitin-proteasome pathway, in modulating antiviral signaling. However, the regulatory mechanisms governing Cactin remain unexplored. In this study, we demonstrate that Usp14 stabilizes Cactin by preventing its ubiquitination and subsequent degradation. Furthermore, Usp14 plays a crucial role in regulating the antiviral function mediated by Cactin. Therefore, our findings elucidate the regulatory mechanism of Cactin in Drosophila, offering a potential target for the prevention and treatment of viral infections.

Tumor-associated macrophage-induced circMRCKα encodes a peptide to promote glycolysis and progression in hepatocellular carcinoma.

The tumor-associated macrophages (TAMs) play multifaceted roles in the progression of hepatocellular carcinoma (HCC). However, the involvement of circular RNAs in the interplay between TAMs and HCC remains unclear. Based on Transwell co-culturing and circular RNA sequencing, this study revealed that TAMs enhanced tumor glycolysis and progression by upregulating circMRCKα in HCC cells. Patients with HCC who exhibited elevated circMRCKα levels presented significantly reduced overall survival and greater cumulative recurrence. Notably, we identified a novel functional peptide of 227 amino acids named circMRCKα-227aa, encoded by circMRCKα. Mechanistically, circMRCKα-227aa bound to USP22 and enhanced its protein level to obstruct HIF-1α degradation via the ubiquitin-proteasome pathway, thereby augmenting HCC glycolysis and progression. In clinical HCC samples, a positive correlation was observed between the expression of circMRCKα and the number of infiltrating CD68+ TAMs and expression of USP22. Furthermore, circMRCKα emerged as an independent prognostic risk factor both individually and in conjunction with CD68+ TAMs and USP22. This study illustrated that circMRCKα-227aa, a novel TAM-induced peptide, promotes tumor glycolysis and progression via USP22 binding and HIF-1α upregulation, suggesting that circMRCKα and TAMs could be combined as therapeutic targets in HCC.

The potential role of CMC1 as an immunometabolic checkpoint in T cell immunity.

T cell immunity is critical for human defensive immune response. Exploring the key molecules during the process provides new targets for T cell-based immunotherapies. CMC1 is a mitochondrial electron transport chain (ETC) complex IV chaperon protein. By establishing in-vitro cell culture system and Cmc1 gene knock out mice, we evaluated the role of CMC1 in T cell activation and differentiation. The B16-OVA tumor model was used to test the possibility of targeting CMC1 for improving T cell anti-tumor immunity. We identified CMC1 as a positive regulator in CD8+T cells activation and terminal differentiation. Meanwhile, we found that CMC1 increasingly expressed in exhausted T (Tex) cells. Genetic lost of Cmc1 inhibits the development of CD8+T cell exhaustion in mice. Instead, deletion of Cmc1 in T cells prompts cells to differentiate into metabolically and functionally quiescent cells with increased memory-like features and tolerance to cell death upon repetitive or prolonged T cell receptor (TCR) stimulation. Further, the in-vitro mechanistic study revealed that environmental lactate enhances CMC1 expression by inducing USP7, mediated stabilization and de-ubiquitination of CMC1 protein, in which a mechanism we propose here that the lactate-enriched tumor microenvironment (TME) drives CD8+TILs dysfunction through CMC1 regulatory effects on T cells. Taken together, our study unraveled the novel role of CMC1 as a T cell regulator and its possibility to be utilized for anti-tumor immunotherapy.