Kidney Tissue Proteome Profiles in Short Versus Long Duration of Delayed Graft Function - A Pilot Study in Donation After Circulatory Death Donors.

Introduction: Delayed graft function (DGF) is often defined as the need for dialysis treatment in the first week after a kidney transplantation. This definition, though readily applicable, is generic and unable to distinguish between "types" of DGF or time needed to recover function that may also significantly affect longer-term outcomes. We aimed to profile biological pathways in donation after circulatory death (DCD) kidney donors that correlate with DGF and different DGF durations. Methods: A total of N = 30 DCD kidney biopsies were selected from the UK Quality in Organ Donation (QUOD) biobank and stratified according to DGF duration (immediate function, IF n = 10; "short-DGF" (1-6 days), SDGF n = 10; "long-DGF" (7-22 days), LDGF n = 10). Samples were matched for donor and recipient demographics and analyzed by label-free quantitative (LFQ) proteomics, yielding identification of N = 3378 proteins. Results: Ingenuity pathway analysis (IPA) on differentially abundant proteins showed that SDGF kidneys presented upregulation of stress response pathways, whereas LDGF presented impaired response to stress, compared to IF. LDGF showed extensive metabolic deficits compared to IF and SDGF. Conclusion: DCD kidneys requiring dialysis only in the first week posttransplant present acute cellular injury at donation, alongside repair pathways upregulation. In contrast, DCD kidneys requiring prolonged dialysis beyond 7 days present minimal metabolic and antioxidant responses, suggesting that current DGF definitions might not be adequate in distinguishing different patterns of injury in donor kidneys contributing to DGF.

HSV-1 employs UL56 to antagonize expression and function of cGAMP channels.

DNA sensing is important for antiviral immunity. The DNA sensor cGAS synthesizes 2'3'-cyclic GMP-AMP (cGAMP), a second messenger that activates STING, which induces innate immunity. cGAMP not only activates STING in the cell where it is produced but cGAMP also transfers to other cells. Transporters, channels, and pores (including SLC19A1, SLC46A2, P2X7, ABCC1, and volume-regulated anion channels (VRACs)) release cGAMP into the extracellular space and/or import cGAMP. We report that infection with multiple human viruses depletes some of these cGAMP conduits. This includes herpes simplex virus 1 (HSV-1) that targets SLC46A2, P2X7, and the VRAC subunits LRRC8A and LRRC8C for degradation. The HSV-1 protein UL56 is necessary and sufficient for these effects that are mediated at least partially by proteasomal turnover. UL56 thereby inhibits cGAMP uptake via VRAC, SLC46A2, and P2X7. Taken together, HSV-1 antagonizes intercellular cGAMP transfer. We propose that this limits innate immunity by reducing cell-to-cell communication via the immunotransmitter cGAMP.

A role for vessel-associated extracellular matrix proteins in multiple sclerosis pathology.

Multiple sclerosis (MS) is unsurpassed for its clinical and pathological hetherogeneity, but the biological determinants of this variability are unknown. HLA-DRB1*15, the main genetic risk factor for MS, influences the severity and distribution of MS pathology. This study set out to unravel the molecular determinants of the heterogeneity of MS pathology in relation to HLA-DRB1*15 status. Shotgun proteomics from a discovery cohort of MS spinal cord samples segregated by HLA-DRB*15 status revealed overexpression of the extracellular matrix (ECM) proteins, biglycan, decorin, and prolargin in HLA-DRB*15-positive cases, adding to established literature on a role of ECM proteins in MS pathology that has heretofore lacked systematic pathological validation. These findings informed a neuropathological characterisation of these proteins in a large autopsy cohort of 41 MS cases (18 HLA-DRB1*15-positive and 23 HLA-DRB1*15-negative), and seven non-neurological controls on motor cortical, cervical and lumbar spinal cord tissue. Biglycan and decorin demonstrate a striking perivascular expression pattern in controls that is reduced in MS (-36.5%, p = 0.036 and - 24.7%, p = 0.039; respectively) in lesional and non-lesional areas. A concomitant increase in diffuse parenchymal accumulation of biglycan and decorin is seen in MS (p = 0.015 and p = 0.001, respectively), particularly in HLA-DRB1*15-positive cases (p = 0.007 and p = 0.046, respectively). Prolargin shows a faint parenchymal pattern in controls that is markedly increased in MS cases where a perivascular deposition pattern is observed (motor cortex +97.5%, p = 0.001; cervical cord +49.1%, p = 0.016). Our findings point to ECM proteins and the vascular interface playing a central role in MS pathology within and outside the plaque area. As ECM proteins are known potent pro-inflammatory molecules, their parenchymal accumulation may contribute to disease severity. This study brings to light novel factors that may contribute to the heterogeneity of the topographical variation of MS pathology.

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms.

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

Oncogenic mutations of KRAS modulate its turnover by the CUL3/LZTR1 E3 ligase complex.

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to ∼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions. Through quantitative proteomics, we demonstrate the presence of known KRAS interactors, including ARAF and LZTR1, which are differentially captured by WT and KRAS mutants. Notably, the KRAS mutations G12D, G13D, and Q61H abrogate their association with LZTR1, thereby affecting turnover. Elucidating the implications of LZTR1-mediated regulation of KRAS protein levels in cancer may offer insights into therapeutic strategies targeting KRAS-driven malignancies.

A novel method to quantify fibrin-fibrin and fibrin-α2-antiplasmin cross-links in thrombi formed from human trauma patient plasma.

BACKGROUND: The widespread use of the antifibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, factor (F)XIIIa catalyzes the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links, which increases clot mechanical strength and resistance to fibrinolysis. OBJECTIVES: Here, we developed a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. METHODS: Fibrinogen alpha (FGA) chain (FGA-FGA), fibrinogen gamma (FGG) chain (FGG-FGG), and FGA-α2AP cross-links were quantified using liquid chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring in paired plasma samples from trauma patients prefibrinogen and postfibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analyzed. RESULTS: The abundance of cross-links was significantly increased in trauma patients postcryo, but not Fg-C transfusion (P < .0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibers, the peak thrombin concentration, and FXIII antigen (P < .05). CONCLUSION: We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach, we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared with Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients.

Dietary n-3 polyunsaturated fatty acids alter the number, fatty acid profile and coagulatory activity of circulating and platelet-derived extracellular vesicles: a randomized, controlled crossover trial.

BACKGROUND: Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs. OBJECTIVES: We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs. METHODS: A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation. RESULTS: n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay. CONCLUSIONS: Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis. CLINICAL TRIAL REGISTRY: This trial was registered at clinicaltrials.gov as NCT03203512.

Protein Profiling of Placental Extracellular Vesicles in Gestational Diabetes Mellitus.

Throughout pregnancy, some degree of insulin resistance is necessary to divert glucose towards the developing foetus. In gestational diabetes mellitus (GDM), insulin resistance is exacerbated in combination with insulin deficiency, causing new-onset maternal hyperglycaemia. The rapid reversal of insulin resistance following delivery strongly implicates the placenta in GDM pathogenesis. In this case-control study, we investigated the proteomic cargo of human syncytiotrophoblast-derived extracellular vesicles (STBEVs), which facilitate maternal-fetal signalling during pregnancy, in a UK-based cohort comprising patients with a gestational age of 38-40 weeks. Medium/large (m/l) and small (s) STBEVs were isolated from GDM (n = 4) and normal (n = 5) placentae using ex vivo dual-lobe perfusion and subjected to mass spectrometry. Bioinformatics were used to identify differentially carried proteins and mechanistic pathways. In m/lSTBEVs, 56 proteins were differently expressed while in sSTBEVs, no proteins reached statistical difference. Differences were also observed in the proteomic cargo between m/lSTBEVs and sSTBEVs, indicating that the two subtypes of STBEVs may have divergent modes of action and downstream effects. In silico functional enrichment analysis of differentially expressed proteins in m/lSTBEVs from GDM and normal pregnancy found positive regulation of cytoskeleton organisation as the most significantly enriched biological process. This work presents the first comparison of two populations of STBEVs' protein cargos (m/l and sSTBEVs) from GDM and normal pregnancy isolated using placenta perfusion. Further investigation of differentially expressed proteins may contribute to an understanding of GDM pathogenesis and the development of novel diagnostic and therapeutic tools.

BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.

Tumor necrosis factor receptor 2 activation elicits sex-specific effects on cortical myelin proteins and functional recovery in a model of multiple sclerosis.

Multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS), predominately affects females compared to males. Tumor necrosis factor (TNF), a pro-inflammatory cytokine, signaling through TNF receptor 1 contributes to inflammatory disease pathogenesis. In contrast, TNF receptor 2 signaling is neuroprotective. Current anti-TNF MS therapies are shown to be detrimental to patients due to pleiotropic effects on both pro- and anti-inflammatory functions. Using a non-pertussis toxin (nPTX) experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice, we systemically administered a TNFR2 agonist (p53-sc-mTNFR2) to investigate behavioral and pathophysiological changes in both female and male mice. Our data shows that TNFR2 activation alleviates motor and sensory symptoms in females. However, in males, the agonist only alleviates sensory symptoms and not motor. nPTX EAE induction in TNFR2 global knockout mice caused exacerbated motor symptoms in females along with an earlier day of onset, but not in males. Our data demonstrates that TNFR2 agonist efficacy is sex-specific for alleviation of motor symptoms, however, it effectively reduces mechanical hypersensitivity in both females and males. Altogether, these data support the therapeutic promise TNFR2 agonism holds as an MS therapeutic and, more broadly, to treat central neuropathic pain.

Data-independent acquisition proteomics of cerebrospinal fluid implicates endoplasmic reticulum and inflammatory mechanisms in amyotrophic lateral sclerosis.

While unbiased proteomics of human cerebrospinal fluid (CSF) has been used successfully to identify biomarkers of amyotrophic lateral sclerosis (ALS), high-abundance proteins mask the presence of lower abundance proteins that may have diagnostic and prognostic value. However, developments in mass spectrometry (MS) proteomic data acquisition methods offer improved protein depth. In this study, MS with library-free data-independent acquisition (DIA) was used to compare the CSF proteome of people with ALS (n = 40), healthy (n = 15) and disease (n = 8) controls. Quantified protein groups were subsequently correlated with clinical variables. Univariate analysis identified 7 proteins, all significantly upregulated in ALS versus healthy controls, and 9 with altered abundance in ALS versus disease controls (FDR < 0.1). Elevated chitotriosidase-1 (CHIT1) was common to both comparisons and was proportional to ALS disability progression rate (Pearson r = 0.41, FDR-adjusted p = 0.035) but not overall survival. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1; upregulated in ALS versus healthy controls) was proportional to disability progression rate (Pearson r = 0.53, FDR-adjusted p = 0.003) and survival (Kaplan Meier log-rank p = 0.013) but not independently in multivariate proportional hazards models. Weighted correlation network analysis was used to identify functionally relevant modules of proteins. One module, enriched for inflammatory functions, was associated with age at symptom onset (Pearson r = 0.58, FDR-adjusted p = 0.005) and survival (Hazard Ratio = 1.78, FDR = 0.065), and a second module, enriched for endoplasmic reticulum proteins, was negatively correlated with disability progression rate (r = -0.42, FDR-adjusted p = 0.109). DIA acquisition methodology therefore strengthened the biomarker candidacy of CHIT1 and UCHL1 in ALS, while additionally highlighted inflammatory and endoplasmic reticulum proteins as novel sources of prognostic biomarkers.

A Hybrid Electrospun-Extruded Polydioxanone Suture for Tendon Tissue Regeneration.

Many surgical tendon repairs fail despite advances in surgical materials and techniques. Tendon repair failure can be partially attributed to the tendon's poor intrinsic healing capacity and the repurposing of sutures from other clinical applications. Electrospun materials show promise as a biological scaffold to support endogenous tendon repair, but their relatively low tensile strength has limited their clinical translation. It is hypothesized that combining electrospun fibers with a material with increased tensile strength may improve the suture's mechanical properties while retaining biophysical cues necessary to encourage cell-mediated repair. This article describes the production of a hybrid electrospun-extruded suture with a sheath of submicron electrospun fibers and a core of melt-extruded fibers. The porosity and tensile strength of this hybrid suture is compared with an electrospun-only braided suture and clinically used sutures Vicryl and polydioxanone (PDS). Bioactivity is assessed by measuring the adsorbed serum proteins on electrospun and melt-extruded filaments using mass spectrometry. Human hamstring tendon fibroblast attachment and proliferation were quantified and compared between the hybrid and control sutures. Combining an electrospun sheath with melt-extruded cores created a hybrid braid with increased tensile strength (70.1 ± 0.3N) compared with an electrospun only suture (12.9 ± 1 N, p < 0.0001). The hybrid suture had a similar force at break to clinical sutures, but lower stiffness and stress. The Young's modulus was 772.6 ± 32 MPa for the hybrid suture, 1693.0 ± 69 MPa for PDS, and 3838.0 ± 132 MPa for Vicryl, p < 0.0001. Hybrid sutures had lower overall porosity than electrospun-only sutures (40 ± 4% and 60 ± 7%, respectively, p = 0.0018) but had a significantly larger overall porosity and average pore diameter compared with surgical sutures. There were similar clusters of adsorbed proteins on electrospun and melt-extruded filaments, which were distinct from PDS. Tendon fibroblast attachment and cell proliferation on hybrid and electrospun sutures were significantly higher than on clinical sutures. This study demonstrated that a bioactive suture with increased tensile strength and lower stiffness could be produced by adding a core of 10 µm melt-extruded fibers to a sheath of electrospun fibers. In contrast to currently used sutures, the hybrid sutures promoted a bioactive response: serum proteins adsorbed, and fibroblasts attached, survived, grew along the sutures, and adopted appropriate morphologies.

Deep topographic proteomics of a human brain tumour.

The spatial organisation of cellular protein expression profiles within tissue determines cellular function and is key to understanding disease pathology. To define molecular phenotypes in the spatial context of tissue, there is a need for unbiased, quantitative technology capable of mapping proteomes within tissue structures. Here, we present a workflow for spatially-resolved, quantitative proteomics of tissue that generates maps of protein abundance across tissue slices derived from a human atypical teratoid-rhabdoid tumour at three spatial resolutions, the highest being 40 µm, to reveal distinct abundance patterns of thousands of proteins. We employ spatially-aware algorithms that do not require prior knowledge of the fine tissue structure to detect proteins and pathways with spatial abundance patterns and correlate proteins in the context of tissue heterogeneity and cellular features such as extracellular matrix or proximity to blood vessels. We identify PYGL, ASPH and CD45 as spatial markers for tumour boundary and reveal immune response-driven, spatially-organised protein networks of the extracellular tumour matrix. Overall, we demonstrate spatially-aware deep proteo-phenotyping of tissue heterogeneity, to re-define understanding tissue biology and pathology at the molecular level.

A multi-platform approach to identify a blood-based host protein signature for distinguishing between bacterial and viral infections in febrile children (PERFORM): a multi-cohort machine learning study.

BACKGROUND: Differentiating between self-resolving viral infections and bacterial infections in children who are febrile is a common challenge, causing difficulties in identifying which individuals require antibiotics. Studying the host response to infection can provide useful insights and can lead to the identification of biomarkers of infection with diagnostic potential. This study aimed to identify host protein biomarkers for future development into an accurate, rapid point-of-care test that can distinguish between bacterial and viral infections, by recruiting children presenting to health-care settings with fever or a history of fever in the previous 72 h. METHODS: In this multi-cohort machine learning study, patient data were taken from EUCLIDS, the Swiss Pediatric Sepsis study, the GENDRES study, and the PERFORM study, which were all based in Europe. We generated three high-dimensional proteomic datasets (SomaScan and two via liquid chromatography tandem mass spectrometry, referred to as MS-A and MS-B) using targeted and untargeted platforms (SomaScan and liquid chromatography mass spectrometry). Protein biomarkers were then shortlisted using differential abundance analysis, feature selection using forward selection-partial least squares (FS-PLS; 100 iterations), along with a literature search. Identified proteins were tested with Luminex and ELISA and iterative FS-PLS was done again (25 iterations) on the Luminex results alone, and the Luminex and ELISA results together. A sparse protein signature for distinguishing between bacterial and viral infections was identified from the selected proteins. The performance of this signature was finally tested using Luminex assays and by calculating disease risk scores. FINDINGS: 376 children provided serum or plasma samples for use in the discovery of protein biomarkers. 79 serum samples were collected for the generation of the SomaScan dataset, 147 plasma samples for the MS-A dataset, and 150 plasma samples for the MS-B dataset. Differential abundance analysis, and the first round of feature selection using FS-PLS identified 35 protein biomarker candidates, of which 13 had commercial ELISA or Luminex tests available. 16 proteins with ELISA or Luminex tests available were identified by literature review. Further evaluation via Luminex and ELISA and the second round of feature selection using FS-PLS revealed a six-protein signature: three of the included proteins are elevated in bacterial infections (SELE, NGAL, and IFN-γ), and three are elevated in viral infections (IL18, NCAM1, and LG3BP). Performance testing of the signature using Luminex assays revealed area under the receiver operating characteristic curve values between 89·4% and 93·6%. INTERPRETATION: This study has led to the identification of a protein signature that could be ultimately developed into a blood-based point-of-care diagnostic test for rapidly diagnosing bacterial and viral infections in febrile children. Such a test has the potential to greatly improve care of children who are febrile, ensuring that the correct individuals receive antibiotics. FUNDING: European Union's Horizon 2020 research and innovation programme, the European Union's Seventh Framework Programme (EUCLIDS), Imperial Biomedical Research Centre of the National Institute for Health Research, the Wellcome Trust and Medical Research Foundation, Instituto de Salud Carlos III, Consorcio Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Grupos de Refeencia Competitiva, Swiss State Secretariat for Education, Research and Innovation.

SARS-CoV-2 mutations affect antigen processing by the proteasome to alter CD8+ T cell responses.

Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design.

GTP Cyclohydrolase Drives Breast Cancer Development and Promotes EMT in an Enzyme-Independent Manner.

GTP cyclohydrolase (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. The catalysis of BH4 biosynthesis is tightly regulated for physiological neurotransmission, inflammation, and vascular tone. Paradoxically, BH4 has emerged as an oncometabolite regulating tumor growth, but the effects on tumor development remain controversial. Here, we found that GCH1 potentiated the growth of triple-negative breast cancer (TNBC) and HER2+ breast cancer and transformed nontumor breast epithelial cells. Independent of BH4 production, GCH1 protein induced epithelial-to-mesenchymal transition by binding to vimentin (Vim), which was mediated by HSP90. Conversely, GCH1 ablation impaired tumor growth, suppressed Vim in TNBC, and inhibited EGFR/ERK signaling while activating the p53 pathway in estrogen receptor-positive tumor cells. GCH1 deficiency increases tumor cell sensitivity to HSP90 inhibition and endocrine treatments. In addition, high GCH1 correlated with poor breast cancer survival. Together, this study reveals an enzyme-independent oncogenic role of GCH1, presenting it as a potential target for therapeutic development. SIGNIFICANCE: GTP cyclohydrolase functions as an oncogene in breast cancer and binds vimentin to induce epithelial-to-mesenchymal transition independently of its enzyme activity, which confers targetable vulnerabilities for developing breast cancer treatment strategies.

Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.

While defective α-synuclein homeostasis is central to Parkinson's pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of α-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.

The TNFR1 antagonist Atrosimab reduces neuronal loss, glial activation and memory deficits in an acute mouse model of neurodegeneration.

Tumor necrosis factor alpha (TNF-α) and its key role in modulating immune responses has been widely recognized as a therapeutic target for inflammatory and neurodegenerative diseases. Even though inhibition of TNF-α is beneficial for the treatment of certain inflammatory diseases, total neutralization of TNF-α largely failed in the treatment of neurodegenerative diseases. TNF-α exerts distinct functions depending on interaction with its two TNF receptors, whereby TNF receptor 1 (TNFR1) is associated with neuroinflammation and apoptosis and TNF receptor 2 (TNFR2) with neuroprotection and immune regulation. Here, we investigated the effect of administering the TNFR1-specific antagonist Atrosimab, as strategy to block TNFR1 signaling while maintaining TNFR2 signaling unaltered, in an acute mouse model for neurodegeneration. In this model, a NMDA-induced lesion that mimics various hallmarks of neurodegenerative diseases, such as memory loss and cell death, was created in the nucleus basalis magnocellularis and Atrosimab or control protein was administered centrally. We showed that Atrosimab attenuated cognitive impairments and reduced neuroinflammation and neuronal cell death. Our results demonstrate that Atrosimab is effective in ameliorating disease symptoms in an acute neurodegenerative mouse model. Altogether, our study indicates that Atrosimab may be a promising candidate for the development of a therapeutic strategy for the treatment of neurodegenerative diseases.

Biallelic FBXW7 knockout induces AKAP8-mediated DNA damage in neighbouring wildtype cells.

Colorectal cancer possesses marked intratumoral heterogeneity. While subclonal interactions between Vogelstein driver mutations have been extensively studied, less is known about competitive or cooperative effects between subclonal populations with other cancer driver mutations. FBXW7 is a cancer driver mutation which is present in close to 17% of colorectal cancer cells. In this study, we generated isogenic FBXW7 mutant cells using CRISPR-Cas9. We identified an upregulation of oxidative phosphorylation and DNA damage in FBXW7 mutant cells, which surprisingly proliferated at a decreased rate compared to wildtype cells. To determine subclonal interactions, wildtype and mutant FBXW7 cells were cocultured using a Transwell system. Wildtype cells cocultured with FBXW7 mutant cells similarly developed DNA damage which was not observed when wildtype cells were co-cultured with other wildtype cells, suggesting that FBXW7 mutant cells were inducing DNA damage in neighbouring wildtype cells. Using mass spectrometry, we identified AKAP8 as being secreted by FBXW7 mutant cells into the coculture media. Furthermore, overexpression of AKAP8 in wildtype cells recapitulated the DNA damage phenotype observed during coculture, while co-culture of wildtype cells with double mutant FBXW7-/-/AKAP8-/- cells abrogated the DNA damage phenotype. Here, we describe a hitherto unknown phenomenon of AKAP8-mediated DNA damage from FBXW7 mutant to neighbouring wildtype cells. Our findings demonstrate the importance of elucidating the local effect of cancer driver mutations between subclonal populations.

Loss of FAM111B protease mutated in hereditary fibrosing poikiloderma negatively regulates telomere length.

Hereditary fibrosing poikiloderma (HFP) is a rare human dominant negative disorder caused by mutations in the FAM111B gene that encodes a nuclear trypsin-like serine protease. HFP patients present with symptoms including skin abnormalities, tendon contractures, myopathy and lung fibrosis. We characterized the cellular roles of human FAM111B using U2OS and MCF7 cell lines and report here that the protease interacts with components of the nuclear pore complex. Loss of FAM111B expression resulted in abnormal nuclear shape and reduced telomeric DNA content suggesting that FAM111B protease is required for normal telomere length; we show that this function is independent of telomerase or recombination driven telomere extension. Even though FAM111B-deficient cells were proficient in DNA repair, they showed hallmarks of genomic instability such as increased levels of micronuclei and ultra-fine DNA bridges. When mutated as in HFP, FAM111B was more frequently localized to the nuclear envelope, suggesting that accumulation of the mutated protease at the nuclear periphery may drive the disease pathology.