The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron microscopy to investigate the ligand-binding pathways to the active site of the enzyme. Each subunit of GDH comprises one hexamer-forming core domain and one nucleotide-binding domain (NAD domain), which spontaneously opens and closes the active-site cleft situated between the two domains. In the presence of NADP, the potential map of GDH hexamer, assuming D3 symmetry, was determined at a resolution of 2.4 Å, but the NAD domain was blurred due to the conformational variety. After focused classification with respect to the NAD domain, the potential maps interpreted as NADP molecules appeared at five different sites in the active-site cleft. The subunits associated with NADP molecules were close to one of the four metastable conformations in the unliganded state. Three of the five binding sites suggested a pathway of NADP molecules to approach the active-site cleft for initiating the enzymatic reaction. The other two binding modes may rarely appear in the presence of glutamate, as demonstrated by the reaction kinetics. Based on the visualized structures and the results from the enzymatic kinetics, we discussed the binding modes of NADP to GDH in the absence and presence of glutamate.

Functional networks in cells are created by physical, genetic, and regulatory interactions. Mapping them and annotating their functions by available methods remains a challenge. We use affinity purification mass spectrometry (AP-MS) coupled with SLiMFinder to discern such a network involving 26S proteasome non-ATPase regulatory subunit 9 (PSMD9), a chaperone of proteasome assembly. Approximately 20% of proteins within the PSMD9 interactome carry a short linear motif (SLiM) of the type 'EXKK'. The binding of purified PSMD9 with the peptide sequence ERKK, proteins heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1; containing ERKK), and peroxiredoxin-6 (PRDX6; containing EAKK) provided proof of principle for this motif-driven network. The EXKK motif in the peptide primarily interacts with the coiled-coil N domain of PSMD9, a unique interaction not reported for any coiled-coil domain. PSMD9 knockout (KO) HEK293 cells experience endoplasmic reticulum (ER) stress and respond by increasing the unfolded protein response (UPR) and reducing the formation of aggresomes and lipid droplets. Trans-expression of PSMD9 in the KO cells rescues lipid droplet formation. Overexpression of PSMD9 in HEK293 cells results in reduced UPR, and increased lipid droplet and aggresome formation. The outcome argues for the prominent role of PSMD9 in maintaining proteostasis. Probable mechanisms involve the binding of PSMD9 to binding immunoglobulin protein (BIP/GRP78; containing EDKK), an endoplasmic reticulum chaperone and key regulator of the UPR, and fatty acid synthase (FASN; containing ELKK), involved in fatty acid synthesis/lipid biogenesis. We propose that PSMD9 acts as a buffer in the cellular milieu by moderating the UPR and enhancing aggresome formation to reduce stress-induced proteotoxicity. Akin to waves created in ponds that perpetuate to a distance, perturbing the levels of PSMD9 would cause ripples down the networks, affecting final reactions in the pathway, one of which is altered proteostasis.

Colorectal cancer (CRC) is one of the most life-threatening neoplasias in terms of incidence and mortality worldwide. Vitamin D deficiency has been associated with an increased risk of CRC. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2 D3 ], the most active vitamin D metabolite, is a pleiotropic hormone that, through its binding to a transcription factor of the nuclear receptor superfamily, is a major regulator of the human genome. 1,25(OH)2 D3 acts on colon carcinoma and stromal cells and displays tumor protective actions. Here, we review the variety of molecular mechanisms underlying the effects of 1,25(OH)2 D3 in CRC, which affect multiple processes that are dysregulated during tumor initiation and progression. Additionally, we discuss the epidemiological data that associate vitamin D deficiency and CRC, and the most relevant randomized controlled trials of vitamin D3 supplementation conducted in both healthy individuals and CRC patients.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin-substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening.

The basic structures of membrane lipids that compose biomembranes differ among species; i.e., in mammals, the primary structure of long-chain base (LCB), the common backbone of ceramides and complex sphingolipids, is sphingosine, whereas, in yeast Saccharomyces cerevisiae, it is phytosphingosine, and S. cerevisiae does not have sphingosine. In addition, the sterol, which is coordinately involved in various functions with complex sphingolipids, is cholesterol in mammals, while in yeast it is ergosterol. Previously, it was found that yeast cells are viable when the structure of LCBs is replaced by sphingosine by supplying an exogenous LCB to cells lacking LCB biosynthesis. Here, we characterized yeast cells having sphingosine instead of phytosphingosine (sphingosine cells). Sphingosine cells exhibited a strong growth defect when biosynthesis of ceramides or complex sphingolipids was inhibited, indicating that, in the sphingosine cells, exogenously added sphingosine is required to be further metabolized. The sphingosine cells exhibited hypersensitivity to various environmental stresses and had abnormal plasma membrane and cell wall properties. Furthermore, we also established a method for simultaneous replacement of both LCB and sterol structures with those of mammals (sphingosine/cholesterol cells). The multiple stress hypersensitivity and abnormal plasma membrane and cell wall properties observed in sphingosine cells were also observed in sphingosine/cholesterol cells, suggesting that simultaneous replacement of both LCB and sterol structures with those of mammals cannot prevent these abnormal phenotypes. This is the first study to our knowledge showing that S. cerevisiae can grow even if LCB and sterol structures are simultaneously replaced with mammalian types.

The recurrent nature of coronavirus outbreaks, severity of the COVID-19 pandemic, rapid emergence of novel variants, and concerns over the effectiveness of existing vaccines against novel variants have highlighted the need to develop therapeutic interventions. Targeted efforts to identify inhibitors of crucial viral proteins are the preferred strategy. In this study, we screened FDA-approved and natural product libraries using in silico approach for potential hits against the SARS-CoV-2 main protease (Mpro) and experimentally validated their potency using in vitro biochemical and cell-based assays. Seven potential hits were identified through in silico screening and were subsequently evaluated in SARS-CoV-2-based cell-free assays, followed by testing in the HCoV-229E-based culture system. Of the tested compounds, 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1-isopropyl-1H-benzofuro[3,2-b]pyrazolo[4,3-e]pyridin-3(2H)-one (PubChem CID:71755304, hereafter referred to as STL522228) exhibited significant antiviral activity. Subsequently, its potential as a novel COVID therapeutic molecule was validated in the SARS-CoV-2-culture system, where STL522228 demonstrated superior antiviral activity (EC50  = 0.44 μm) compared to Remdesivir (EC50  = 0.62 μm). Based on these findings, we report the strong anti-coronavirus activity of STL522228, and propose that it as a promising pan-coronavirus Mpro inhibitor for further experimental and preclinical validation.

As a known receptor-ligand pair for mediating cell-cell or cell-extracellular matrix adhesions, cluster of differentiation 44 (CD44)-hyaluronan (HA) interactions are not only determined by molecular weight (MW) diversity of HA, but also are regulated by external physical or mechanical factors. However, the coupling effects of HA MW and shear flow are still unclear. Here, we compared the differences between high molecular weight HA (HHA) and low molecular weight HA (LHA) binding to CD44 under varied shear stresses. The results demonstrated that HHA dominated the binding phase but LHA was in favour of the shear resistance phase, respectively, under shear stress range ≤ 1.0 dyne·cm-2 . This difference was attributed to the high binding strength of the CD44-HHA interaction, as well as the optimal distribution matching between both CD44 and HA sides. Activation of the intracellular signal pathway was sensitive to both HA MW and shear flow. Our findings also indicate that only CD44-HHA interaction under shear stress of 0.2 dyne·cm-2 could significantly enhance the clustering of CD44, as well as induce the increase in both CD44 and CD18 expression. The present study offers the basis for further quantification of the features of CD44-HA interactions and their biological functions.

Alternative therapeutic options targeting urologic malignancies, such as germ cell tumours, as well as urothelial, renal and prostate carcinomas, are still urgently needed. The membrane protein CD24 represents a promising immunotherapeutical approach. The present study aimed to decipher the molecular function of CD24 in vitro and evaluate the cytotoxic capacity of a third-generation natural killer (NK) cell chimeric antigen receptor (CAR) against CD24 in urologic tumour cell lines. Up to 20 urologic tumour cell lines and several non-malignant control cells were included. XTT viability assays and annexin V/propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co-immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of CD24 expression by SOX2. N- and O-glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. The study demonstrates that SOX2 transactivates CD24 expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding and post-translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumour cells with NK-CD24-CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24+ tumour cells. Limitations of the study include the in vitro setting, which still has to be confirmed in vivo. In conclusion, we show that CD24 is a promising novel target for immune therapeutic approaches targeting urologic malignancies.

FLAD1, along with its FAD synthase (FADS, EC 2.7.7.2) product, is crucial for flavin homeostasis and, due to its role in the mitochondrial respiratory chain and nuclear epigenetics, is closely related to cellular metabolism. Therefore, it is not surprising that it could be correlated with cancer. To our knowledge, no previous study has investigated FLAD1 prognostic significance in pancreatic ductal adenocarcinoma (PDAC). Thus, in the present work, the FAD synthesis process was evaluated in two PDAC cell lines: (a) PANC-1- and PANC-1-derived cancer stem cells (CSCs), presenting the R273H mutation in the oncosuppressor p53, and (b) MiaPaca2 and MiaPaca2-derived CSCs, presenting the R248W mutation in p53. As a control, HPDE cells expressing wt-p53 were used. FADS expression/activity increase was found with malignancy and even more with stemness. An increased FAD synthesis rate in cancer cell lines is presumably demanded by the increase in the FAD-dependent lysine demethylase 1 protein amount as well as by the increased expression levels of the flavoprotein subunit of complex II of the mitochondrial respiratory chain, namely succinate dehydrogenase. With the aim of proposing FADS as a novel target for cancer therapy, the inhibitory effect of Chicago Sky Blue on FADS enzymatic activity was tested on the recombinant 6His-hFADS2 (IC50  = 1.2 μm) and PANC-1-derived CSCs' lysate (IC50  = 2-10 μm). This molecule was found effective in inhibiting the growth of PANC-1 and even more of its derived CSC line, thus assessing its role as a potential chemotherapeutic drug.

Lori Passmore is a Group Leader at the MRC Laboratory of Molecular Biology (MRC-LMB). She studied Biochemistry at the University of British Columbia in Vancouver (Canada), before moving to the UK in 1999 for a PhD at the Institute of Cancer Research. After completing her PhD, Lori moved to Cambridge, where she became a Post-Doctoral Fellow at the MRC-LMB. In 2009, Lori started her own group at the MRC-LMB and was subsequently awarded an ERC Starting Grant (2011), an ERC Consolidator Grant (2017) and a Wellcome Discovery Award (2023). She was also elected into the EMBO Young Investigator Programme (2015) and EMBO Membership (2018). Lori's research focusses on the determination of the structures of protein complexes that regulate gene expression, using primarily cryo-electron microscopy and in vitro assays. Her work has contributed significantly to our understanding of the underlying molecular mechanisms of cellular processes, giving insights into human physiology and disease. In this interview, Lori provides an overview of her research and discusses current challenges in the field, recalls the key events and collaborations that have helped shape her successful research career and offers advice to early career scientists.

Magnesium (Mg2+ ) is the most abundant divalent cation in cells and plays key roles in almost all biological processes. CBS-pair domain divalent metal cation transport mediators (CNNMs) are a newly characterized class of Mg2+ transporters present throughout biology. Originally discovered in bacteria, there are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer. Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a cystathionine-β-synthase (CBS)-pair domain, and a cyclic nucleotide-binding homology domain. The transmembrane and CBS-pair core are the defining features of CNNM proteins with over 20 000 protein sequences known from over 8000 species. Here, we review the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of their regulation and mechanism of ion transport. Recent structures of prokaryotic CNNMs confirm the transmembrane domain mediates ion transport with the CBS-pair domain likely playing a regulatory role through binding divalent cations. Studies of mammalian CNNMs have identified new binding partners. These advances are driving progress in understanding this deeply conserved and widespread family of ion transporters.

As a self-degrading and highly conserved survival mechanism, autophagy plays an important role in maintaining cell survival and recycling. The discovery of autophagy-related (ATG) genes has revolutionized our understanding of autophagy. Lysosomal membrane proteins (LMPs) are important executors of lysosomal function, and increasing evidence has demonstrated their role in the induction and regulation of autophagy. In addition, the functional dysregulation of the process mediated by LMPs at all stages of autophagy is closely related to neurodegenerative diseases and cancer. Here, we review the role of LMPs in autophagy, focusing on their roles in vesicle nucleation, vesicle elongation and completion, the fusion of autophagosomes and lysosomes, and degradation, as well as their broad association with related diseases.

The 'A Disintegrin And Metalloproteinase 10' (ADAM10) has gained considerable attention due to its discovery as an 'α-secretase' involved in the nonamyloidogenic processing of the amyloid precursor protein, thereby possibly preventing the excessive generation of the amyloid beta peptide, which is associated with the pathogenesis of Alzheimer's disease. ADAM10 was found to exert many additional functions, cleaving about 100 different membrane proteins. ADAM10 is involved in many pathophysiological conditions, ranging from cancer and autoimmune disorders to neurodegeneration and inflammation. ADAM10 cleaves its substrates close to the plasma membrane, a process referred to as ectodomain shedding. This is a central step in the modulation of the functions of cell adhesion proteins and cell surface receptors. ADAM10 activity is controlled by transcriptional and post-translational events. The interaction of ADAM10 with tetraspanins and the way they functionally and structurally depend on each other is another topic of interest. In this review, we will summarize findings on how ADAM10 is regulated and what is known about the biology of the protease. We will focus on novel aspects of the molecular biology and pathophysiology of ADAM10 that were previously poorly covered, such as the role of ADAM10 on extracellular vesicles, its contribution to virus entry, and its involvement in cardiac disease, cancer, inflammation, and immune regulation. ADAM10 has emerged as a regulator controlling cell surface proteins during development and in adult life. Its involvement in disease states suggests that ADAM10 may be exploited as a therapeutic target to treat conditions associated with a dysfunctional proteolytic activity.

Amino acids act as versatile nutrients driving cell growth and survival, especially in cancer cells. Amino acid metabolism comprises numerous metabolic networks and is closely linked with intracellular redox balance and epigenetic regulation. Reprogrammed amino acid metabolism has been recognized as a ubiquitous feature in tumour cells. This review outlines the metabolism of several primary amino acids in cancer cells and highlights the pivotal role of amino acid metabolism in sustaining redox homeostasis and regulating epigenetic modification in response to oxidative and genetic stress in cancer cells.

Recent discoveries have highlighted the prevalence of cotranslational assembly in proteomes, revealing a range of mechanisms that enables the assembly of protein complex subunits on the ribosome. Structural analyses have uncovered emergent properties that may inherently control whether a subunit undergoes cotranslational assembly. However, the evolutionary paths that have yielded such complexes over an extended timescale remain largely unclear. In this review, we reflect on historical experiments that contributed to the field, including breakthroughs that have made possible the proteome-wide detection of cotranslational assembly, and the technical challenges yet to be overcome. We introduce a simple framework that encapsulates the hallmarks of cotranslational assembly and discuss how results from new experiments are shaping our view of the mechanistic, structural and evolutionary factors driving the phenomenon.

Notch pathway is an evolutionarily conserved signalling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. Notch signalling plays important roles in many developmental processes, making it difficult to name a tissue or a developing organ that does not depend on Notch function at one stage or another. Thus, dysregulation of Notch signalling is associated with many developmental defects and various pathological conditions, including cancer. Although many recent advances have been made to reveal different aspects of the Notch signalling mechanism and its intricate regulation, there are still many unanswered questions related to how the Notch signalling pathway functions in so many developmental events. The same pathway can be deployed in numerous cellular contexts to play varied and critical roles in an organism's development and this is only possible because of the complex regulatory mechanisms of the pathway. In this review, we provide an overview of the mechanism and regulation of the Notch signalling pathway along with its multifaceted functions in different aspects of development and disease.

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V-ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin-1. By utilizing the whole-endolysosome patch-clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten-of-second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2-GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH-buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome-associated membrane protein 1 (LAMP1). We propose that the TMEM175-mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson's disease and lysosome storage disorders.

Ocular diseases are a highly heterogeneous group of phenotypes, caused by a spectrum of genetic variants and environmental factors that exhibit diverse clinical symptoms. As a result of its anatomical location, structure and immune privilege, the eye is an ideal system to assess and validate novel genetic therapies. Advances in genome editing have revolutionized the field of biomedical science, enabling researchers to understand the biology behind disease mechanisms and allow the treatment of several health conditions, including ocular pathologies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing facilitates efficient and specific genetic modifications in the nucleic acid sequence, resulting in permanent changes at the genomic level. This approach has advantages over other treatment strategies and is promising for the treatment of various genetic and non-genetic ocular conditions. This review provides an overview of the CRISPR/CRISPR-associated protein 9 (Cas9) system and summarizes recent advances in the therapeutic application of CRISPR/Cas9 for the treatment of various ocular pathologies, as well as future challenges.

Cancer initiation and progression heavily rely on microenvironmental cues derived from various components of the niche including the extracellular matrix (ECM). ECM is a complex macromolecular network that governs cell functionality. Although the two-dimensional (2D) cell culture systems provide useful information at the molecular level and preclinical testing, they could not accurately represent the in vivo matrix microenvironmental architecture. Hence, it is no surprise that researchers in the last decade have focussed their efforts on establishing novel advanced in vitro culture models that mimic tumour and tissue-specific niches and interactions. These numerous three-dimensional (3D) culture systems that are now widely available, as well as those still under development, grant researchers with new, improved tools to study cancer progression and to explore innovative therapeutic options. Herein, we report on the emerging methods and cutting-edge technologies in 3D cell culture platforms and discuss their potential use in unveiling tumour microenvironmental cues, drug screening and personalized treatment.