The immune landscape varies among individuals. It determines the immune response and results in surprisingly diverse symptoms, even in response to similar external stimuli. However, the detailed mechanisms underlying such diverse immune responses have remained mostly elusive. The utilization of recently developed single-cell multimodal analysis platforms has started to answer this question. Emerging studies have elucidated several molecular networks that may explain diversity with respect to age or other factors. An elaborate interplay between inherent physical conditions and environmental conditions has been demonstrated. Furthermore, the importance of modifications by the epigenome resulting in transcriptome variation among individuals is gradually being revealed. Accordingly, epigenomes and transcriptomes are direct indicators of the medical history and dynamic interactions with environmental factors. Coronavirus disease 2019 (COVID-19) has recently become one of the most remarkable examples of the necessity of in-depth analyses of diverse responses with respect to various factors to improve treatment in severe cases and to prevent viral transmission from asymptomatic carriers. In fact, determining why some patients develop serious symptoms is still a pressing issue. Here, we review the current "state of the art" in single-cell analytical technologies and their broad applications to healthy individuals and representative diseases, including COVID-19.

α-Tocopheryl succinate (TS), a redox-silent succinyl ester of natural α-Tocopherol, has emerged as a novel anti-cancer agent. However, the underlying mechanism is unclear. We found that the terminal dicarboxylic moiety of tocopheryl esters contributes to apoptosis induction and thus cytotoxicity. To further examine this relationship, we compared the pro-apoptotic activity of TS, which has four carbon atoms in the terminal dicarboxylic moiety, to that of a newly synthesized, tocopheryl glutarate (Tglu), which has five. Cytotoxicity assays in vitro confirmed that TS stimulated apoptosis, while Tglu was non-cytotoxic. In investigating biological mechanisms leading to these opposing effects, we found that TS caused an elevation of intracellular superoxide, but Tglu did not. TS increased intracellular Ca2+ in cultured cells, suggesting induction of endoplasmic reticulum (ER) stress; however, Tglu did not affect Ca2+ homeostasis. 1,4,5-trisphosphate (IP3 ) receptor antagonist 2-Aminoethyl diphenylborinate (2-APB) decreased TS-induced intracellular Ca2+ , restored mitochondrial activity and cell viability in TS-treated cells, establishing the ER-mitochondria relationship in apoptosis induction. Moreover, real-time PCR, immunostaining and Western blotting assays revealed that TS downregulated glucose-regulated protein 78 (GRP78), which maintains ER homeostasis and promotes cell survival. Conversely, Tglu upregulates GRP78. Taken together, our results suggest a model in which TS-mediated superoxide production and GRP78 inhibition induce ER stress, which elevates intracellular Ca2+ and depolarizes mitochondria, leading to apoptosis. Because Tglu does not affect superoxide generation and increases GRP78 expression, it inhibits ER stress and is thereby non-cytotoxic. Our research provides insight into the structure-activity relationship of tocopheryl esters regarding the induction of apoptosis.

Bacterial biofilms consist of bacterial cells embedded within a self-produced extracellular polymeric substance (EPS) composed of exopolysaccharides, extra cellular DNA, proteins and lipids. The enzyme Dispersin B (DspB) is a CAZy type 20 β-hexosaminidase enzyme that catalyses the hydrolysis of poly-N-acetylglucosamine (PNAG), a major biofilm polysaccharide produced by a wide variety of biofilm-forming bacteria. Native PNAG is partially de-N-acetylated, and the degree of deacetylation varies between species and dependent on the environment. We have previously shown that DspB is able to perform both endo- and exo-glycosidic bond cleavage of PNAG depending on the de-N-acetylation patterns present in the PNAG substrate. Here, we used a combination of synthetic PNAG substrate analogues, site-directed mutagenesis and in vitro biofilm dispersal assay to investigate the molecular basis for the endo-glycosidic cleavage activity of DspB and the importance of this activity for dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. We found that D242 contributes to the endoglycosidase activity of DspB through electrostatic interactions with cationic substrates in the -2 binding site. A DspBD242N mutant was highly deficient in endoglycosidase activity while maintaining exoglycosidase activity. When used to disperse S. epidermidis biofilms, this DspBD242N mutant resulted in an increase in residual biofilm biomass after treatment when compared to wild-type DspB. These results suggest that the de-N-acetylation of PNAG in S. epidermidis biofilms is not uniformly distributed and that the endoglycosidase activity of DspB is required for efficient biofilm dispersal.

The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.

The expression of CCAAT/enhancer-binding protein (C/EBP) family members and peroxisome proliferator-activated receptor γ (PPAR γ) is essential for the differentiation of pre-adipocyte 3T3-L1 cells into mature adipocytes induced by a combined stimulation with dexamethasone, 3-isobutyl-1-methylxanthine and insulin (DMI). We herein demonstrated that the RNA helicase DDX5, the expression of which was induced by DMI, played an important role in the adipocyte differentiation of 3T3-L1 cells. The DMI-induced accumulation of lipid droplets and expression of adipocyte markers in 3T3-L1 cells were significantly inhibited by the knockdown of DDX5. The knockdown of DDX5 interfered with the expressional induction of C/EBPδ, which was the first to be induced in the transcription factor cascade, and inhibited the subsequent expression of the other transcription factors, C/EBPβ, PPARγ and C/EBPα. DDX5 interacted with the glucocorticoid receptor (GR), which induced the expression of C/EBPδ. The knockdown of DDX5 failed to induce the nuclear translocation of GR, suggesting the essential role of DDX5 in the early stage of adipocyte differentiation. Furthermore, the reconstitution of DDX5, but not the DDX5 mutant (K144N) lacking RNA helicase activity, restored DMI-induced GR activation and adipocyte differentiation in 3T3-L1 cells in which DDX5 was knocked down, confirming that the RNA helicase activity of DDX5 is essential for adipogenesis. Collectively, these results revealed for the first time that DDX5 is necessary for GR activation and plays an essential role in early adipocyte differentiation.

Voltage-gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage-gated proton channels (HV ) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV 1, AcHV 2, and AcHV 3) are distributed throughout the whole animal. Patch-clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV 1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV 2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV 3, which differs from AcHV 1 and AcHV 2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species.

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.

Lung cancer is the most aggressive cancer with the highest mortality and incidence rates worldwide. MicroRNAs have been identified as potential targets for non-small cell lung cancer (NSCLC) treatment. However, the modulatory role of miR-514b-5p in NSCLC progression is little known. In the present study, miRNA expression datasets for NSCLC were downloaded from the Cancer Genome Atlas and Gene Ontology Omnibus databases. Gene expression was assessed using a quantitative real-time PCR, and western blot analysis and immunohistochemical staining was used to determine protein expression. Gain and loss of function experiments were performed to investigate the impact of miR-514b-5p and small glutamine-rich tetratricopeptide repeat-containing protein beta (SGTB) on cell proliferation and apoptosis. RNA immunoprecipitation and dual-luciferase assays were performed to analyse the target gene of miR-514b-5p. The biological roles of miR-514b-5p were lastly evaluated using nude mouse tumorigenicity assays in vivo. We found that miR-514b-5p was dramatically increased in NSCLC tissues and higher miR-514b-5p expression was associated with poorer overall survival in NSCLC patients. Furthermore, overexpression of miR-514b-5p promoted NSCLC cell growth and suppressed apoptosis by inducing the activation of the phosphatidylinositol-3-kinase (PI3K)/AKT and p38 signalling pathways. Mechanistically, dual-luciferase and the RNA immunoprecipitation results highlighted that SGTB was a target gene of miR-514b-5p. Moreover, overexpression of SGTB reduced cell division and promoted apoptosis in vitro through blocking the PI3K/AKT and p38 signalling pathways. Our findings indicated that miR-514b-5p contributes to carcinoma progression in NSCLC via the PI3K/AKT and p38 signalling pathways by targeting SGTB and this could be a promising diagnostic and therapeutic target for the treatment of NSCLC.

Succinic semialdehyde dehydrogenase (SSADH) catalyses the conversion of succinic semialdehyde into succinic acid and two electrons are transferred to NAD(P)+ to yield NAD(P)H. Our previous work has already reported the catalytic role of Cys289 of two-cysteine SSADH from Acinetobacter baumannii (AbSSADH). However, the mechanistic role of the neighbouring conserved Cys291 and Glu255 remains unexplored. In this study, the functional roles of Cys291 and Glu255 in AbSSADH catalysis have been characterized. Results demonstrated that the E255A activity was almost completely lost, ~ 7000-fold lower than the wild-type (WT), indicating that Glu255 is very crucial and directly involved in AbSSADH catalysis. However, the C291A and C291S variants activity and catalytic turnover (kcat ) decreased ~ 2-fold and 9-fold respectively. To further characterize the functional roles of Cys291, we employed two pH-dependent methods; pre-steady-state burst amplitude and NADP-enzyme adduct formation. The results showed that the pKa values of catalytic Cys289 measured for the WT and C291A reactions were 7.8 and 8.7-8.8, respectively, suggesting that Cys291 can lower the pKa of Cys289 and consequently trigger the deprotonation of a Cys289 thiol. In addition, the Cys291 also plays a role in disulfide/sulfhydryl redox regulation for AbSSADH activity. Hence, we demonstrated for the first time the dual functions of Cys291 in enhancing the nucleophilicity of the catalytic Cys289 and regulating a disulfide/sulfhydryl redox switch for AbSSADH catalysis. The mechanistic insights into the nucleophilicity enhancement of the catalytic cysteine of AbSSADH might be applicable to understanding how the microenvironment increases cysteine reactivity in other enzymes in the aldehyde dehydrogenase superfamily.

The SID1 transmembrane family, member 2, namely, Sidt2, is a highly glycosylated multichannel lysosomal transmembrane protein, but its specific physiological function remains unknown. Lysosomal membrane proteins are very important for the executive functioning of lysosomes. As an important part of the lysosomal membrane, Sidt2 can maintain the normal morphology of lysosomes and help stabilize them from the acidic pH environment within. As a receptor/transporter, it binds and transports nucleic acids and mediates the uptake and degradation of RNA and DNA by the lysosome. During glucose metabolism, deletion of Sidt2 can cause an increase in fasting blood glucose and the impairment of grape tolerance, which is closely related to the secretion of insulin. During lipid metabolism, the loss of Sidt2 can cause hepatic steatosis and lipid metabolism disorders and can also play a role in signal regulation and transport. Here, we review the function of the lysosomal membrane protein Sidt2, and focus on its role in glucose and lipid metabolism, autophagy and nucleotide (DNA/RNA) transport.

Brain health requires circuits, cells and molecular pathways to adapt when challenged and to promptly reset once the challenge has resolved. Neurodegeneration occurs when adaptability becomes confined, causing challenges to overwhelm neural circuitry. Studies of rare and common neurodegenerative diseases suggest that the accumulation of lipids can compromise circuit adaptability. Using microglia as an example, we review data that suggest increased lipid concentrations cause dysfunctional inflammatory responses to immune challenges, leading to Alzheimer's disease, Parkinson's disease and dementia. We highlight current approaches to treat lipid metabolic and clearance pathways and identify knowledge gaps towards restoring adaptive homeostasis in individuals who are at-risk of losing cognition.

Sarcopenia is a common disorder that leads to a progressive decrease in skeletal muscle function in elderly people. Exercise effectively prevents or delays the onset and progression of sarcopenia. However, the molecular mechanisms underlying how exercise intervention improves skeletal muscle atrophy remain unclear. In this study, we found that 21-month-old zebrafish had a decreased swimming ability, reduced muscle fibre cross-sectional area, unbalanced protein synthesis, and degradation, increased oxidative stress, and mitochondrial dysfunction, which suggests zebrafish are a valuable model for sarcopenia. Eight weeks of exercise intervention attenuated these pathological changes in sarcopenia zebrafish. Moreover, the effects of exercise on mitochondrial dysfunction were associated with the activation of the AMPK/SIRT1/PGC-1α axis and 15-PGDH downregulation. Our results reveal potential therapeutic targets and indicators to treat age-related sarcopenia using exercise intervention.

Cancer immunotherapies emerge as promising strategies for restricting tumour growth. The tumour microenvironment (TME) has a major impact on the anti-tumour immune response and on the efficacy of the immunotherapies. Recent studies have linked changes in the ambient temperature with particular immuno-metabolic reprogramming and anti-cancer immune response in laboratory animals. Here, we describe the energetic balance of the organism during change in temperature, and link this to the immune alterations that could be of relevance for cancer, as well as for other human diseases. We highlight the contribution of the gut microbiota in modifying this interaction. We describe the overall metabolic response and underlying mechanisms of tumourigenesis in mouse models at varying ambient temperatures and shed light on their potential importance in developing therapeutics against cancer.

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.

Su-(var)3-9 homologue 5 (SUVH5), a member of SUVH family of histone lysine methyltransferase (HKMT) in Arabidopsis, is involved in epigenetic regulation of chromatin by recognizing 5-methyl-cytosine (5mC), in both CpG and non-CpG DNA context, through SRA domain and simultaneously performing the di-methylation of lysine 9 of histone H3 (H3K9) through SET domain. Here, we establish that the SET domain of SUVH5 allosterically restricts the SRA domain to the 5mC containing strand(s) of fully methylated CpG, hemi-methylated CpG and methylated CpHpH DNA. In addition, SET domain enhances the binding affinity of the SRA-SET dual domains to fully-mCpG but not to hemi-mCpG. Also, the recognition of methylated DNA by the SRA positively influences the recognition of H3K9 by the SET domain. Our further studies revealed that the SET domain recognizes the "A(R/K)KST" motif present in H3K9 and in other histone H2A variants. Further, computational analyses and quantum mechanics/molecular mechanics calculations explain the bases for robust mono-MTase but weak di-MTase activities of SUVH5. Given that the majority of eukaryotic proteins, including those involved in epigenetic gene regulation, contain more than one domain, our study suggests that understanding the allosteric regulation among multiple domains of proteins is relevant for unravelling biological outcomes.

Ionotropic glutamate receptors are ligand-gated ion channels essential for fast excitatory neurotransmission in the brain. In contrast to most other members of the iGluR family, the subfamily of delta receptors, GluD1 and GluD2, does not bind glutamate but glycine/D-serine. GluD1 is widely expressed in the brain and the inner ear, where it is required for high-frequency hearing. Furthermore, it has been associated with schizophrenia, autism and depression. X-ray structures of the ligand-binding domain (LBD) of GluD2 have been published; however, no high-resolution structure is available for the ligand-binding domain of GluD1 (GluD1-LBD). Here, we report the X-ray crystal structure of the GluD1-LBD in its apo form at 2.57 Å resolution. Using isothermal titration calorimetry, we show that D-serine binds to the GluD1-LBD in an exothermic manner with a Kd of 160 μm, which is approximately five-fold greater than at GluD2. Furthermore, we identify Glu822 as a critical determinant of receptor activation in GluD1 A654T. In contrast to studies on the GluD2 lurcher mutant A654T, we did not observe any effect of 1 mm D-serine on the spontaneous currents at mouse GluD1 A654T by electrophysiological recordings of Xenopus laevis oocytes as previously also reported by others. These results point towards differences in the structure and dynamics between GluD1 and GluD2. Molecular dynamics simulations were employed to address this observation, suggesting that the apo structure of GluD1 is less flexible than the apo structure of GluD2 and that Pro725 in GluD1 may affect the interlobe closure of the ligand-binding domain of GluD1.

The strong regenerative ability of the liver safeguards the crucial hepatic functions. The balance between hepatocyte proliferation and death is critical for restoring liver size and physiology. Tumour necrosis factor (TNF) alpha-induced protein 8-like 1 (TIPE1) is highly expressed in liver and has been identified as a candidate regulator for cell proliferation and death, being involved in a variety of biological processes and diseases. However, the role of TIPE1 in liver regeneration remains unexplored. In the present study, we found that TIPE1 expression was elevated in the regenerating liver induced by either partial hepatectomy or 10% carbon tetrachloride administration. Mice with hepatocyte conditional Tipe1 knockout presented significantly impaired liver regeneration. Mechanistically, hepatic Tipe1 deficiency decreased the level of reactive oxygen species in hepatocytes, which in turn led to the inhibition of Forkhead box O1 acetylation and microtubule-associated protein 1 light chain 3 I to microtubule-associated protein 1 light chain 3 II conversion, and the accumulation of sequestosome 1. By contrast, forced expression of TIPE1 in hepatocyte significantly promoted liver regeneration following 70% partial hepatectomy and enhanced hepatocyte reactive oxygen species/acetylated-Forkhead box O1 level and autophagy. These findings indicate that TIPE1 plays a crucial role in liver regeneration by finely regulating the oxidative stress and autophagy and is a potential target for medical intervention of liver regeneration.

Tripartite motif-containing protein 27 (TRIM27/also called RFP) is a multifunctional ubiquitin E3 ligase involved in numerous cellular functions, such as proliferation, apoptosis, regulation of the NF-kB pathway, endosomal recycling and the innate immune response. TRIM27 interacts directly with TANK-binding kinase 1 (TBK1) and regulates its stability. TBK1 in complex with autophagy receptors is recruited to ubiquitin chains assembled on the mitochondrial outer membrane promoting mitophagy. Here, we identify TRIM27 as an autophagy substrate, depending on ATG7, ATG9 and autophagy receptors for its lysosomal degradation. We show that TRIM27 forms ubiquitylated cytoplasmic bodies that co-localize with autophagy receptors. Surprisingly, we observed that induced expression of EGFP-TRIM27 in HEK293 FlpIn TRIM27 knockout cells mediates mitochondrial clustering. TRIM27 interacts with autophagy receptor SQSTM1/p62, and the TRIM27-mediated mitochondrial clustering is facilitated by SQSTM/p62. We show that phosphorylated TBK1 is recruited to the clustered mitochondria. Moreover, induced mitophagy activity is reduced in HEK293 FlpIn TRIM27 knockout cells, while re-introduction of EGFP-TRIM27 completely restores the mitophagy activity. Inhibition of TBK1 reduces mitophagy in HEK293 FlpIn cells and in the reconstituted EGFP-TRIM27-expressing cells, but not in HEK293 FlpIn TRIM27 knockout cells. Altogether, these data reveal novel roles for TRIM27 in mitophagy, facilitating mitochondrial clustering via SQSTM1/p62 and mitophagy via stabilization of phosphorylated TBK1 on mitochondria.

Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though β1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.

Decapping is the enzymatic removal of 5' cap structures from mRNAs in eukaryotic cells. Cap structures normally enhance mRNA translation and stability, and their excision commits an mRNA to complete 5'-3' exoribonucleolytic digestion and generally ends the physical and functional cellular presence of the mRNA. Decapping plays a pivotal role in eukaryotic cytoplasmic mRNA turnover and is a critical and highly regulated event in multiple 5'-3' mRNA decay pathways, including general 5'-3' decay, nonsense-mediated mRNA decay (NMD), AU-rich element-mediated mRNA decay, microRNA-mediated gene silencing, and targeted transcript-specific mRNA decay. In the yeast Saccharomyces cerevisiae, mRNA decapping is carried out by a single Dcp1-Dcp2 decapping enzyme in concert with the accessory activities of specific regulators commonly known as decapping activators or enhancers. These regulatory proteins include the general decapping activators Edc1, 2, and 3, Dhh1, Scd6, Pat1, and the Lsm1-7 complex, as well as the NMD-specific factors, Upf1, 2, and 3. Here, we focus on in vivo mRNA decapping regulation in yeast. We summarize recently uncovered molecular mechanisms that control selective targeting of the yeast decapping enzyme and discuss new roles for specific decapping activators in controlling decapping enzyme targeting, assembly of target-specific decapping complexes, and the monitoring of mRNA translation. Further, we discuss the kinetic contribution of mRNA decapping for overall decay of different substrate mRNAs and highlight experimental evidence pointing to the functional coordination and physical coupling between events in mRNA deadenylation, decapping, and 5'-3' exoribonucleolytic decay.

Signal peptide peptidase (SPP) and SPP-like (SPPL) aspartyl intramembrane proteases are known to contribute to sequential processing of type II-oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER-resident family members SPP and SPPL2c were shown to also cleave tail-anchored proteins, including selected SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co-expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail-anchored proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1-4 levels in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1-4 in a cell type- and tissue-dependent manner, identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1-4 by initiating the degradation of these proteins, which might impact cellular trafficking.