SnapShot: Lysosomal Functions.

In addition to their well-defined recycling function, lysosomes act as metabolic signaling hubs that adjust cellular metabolism according to the availability of nutrients and growth factors by regulating metabolic kinases and transcription factors on their surface. Moreover, lysosomal hydrolases and ions released to cytosol or extracellular space have recently emerged as important regulators of various cellular processes from cell death to cell division. To view this SnapShot, open or download the PDF.

Interpersonal Gut Microbiome Variation Drives Susceptibility and Resistance to Cholera Infection.

The gut microbiome is the resident microbial community of the gastrointestinal tract. This community is highly diverse, but how microbial diversity confers resistance or susceptibility to intestinal pathogens is poorly understood. Using transplantation of human microbiomes into several animal models of infection, we show that key microbiome species shape the chemical environment of the gut through the activity of the enzyme bile salt hydrolase. The activity of this enzyme reduced colonization by the major human diarrheal pathogen Vibrio cholerae by degrading the bile salt taurocholate that activates the expression of virulence genes. The absence of these functions and species permits increased infection loads on a personal microbiome-specific basis. These findings suggest new targets for individualized preventative strategies of V. cholerae infection through modulating the structure and function of the gut microbiome.

Somatic Mutations Increase Hepatic Clonal Fitness and Regeneration in Chronic Liver Disease.

Normal tissues accumulate genetic changes with age, but it is unknown if somatic mutations promote clonal expansion of non-malignant cells in the setting of chronic degenerative diseases. Exome sequencing of diseased liver samples from 82 patients revealed a complex mutational landscape in cirrhosis. Additional ultra-deep sequencing identified recurrent mutations in PKD1, PPARGC1B, KMT2D, and ARID1A. The number and size of mutant clones increased as a function of fibrosis stage and tissue damage. To interrogate the functional impact of mutated genes, a pooled in vivo CRISPR screening approach was established. In agreement with sequencing results, examination of 147 genes again revealed that loss of Pkd1, Kmt2d, and Arid1a promoted clonal expansion. Conditional heterozygous deletion of these genes in mice was also hepatoprotective in injury assays. Pre-malignant somatic alterations are often viewed through the lens of cancer, but we show that mutations can promote regeneration, likely independent of carcinogenesis.

Lysozyme Counteracts ß-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

Electric Fields and Enzyme Catalysis.

What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme's active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.

Structure of a Pancreatic ATP-Sensitive Potassium Channel.

ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closed conformation. In another structural population, a putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.

Ligand and Target Discovery by Fragment-Based Screening in Human Cells.

Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration.

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Small RNA-mediated activation of sugar phosphatase mRNA regulates glucose homeostasis.

Glucose homeostasis is strictly controlled in all domains of life. Bacteria that are unable to balance intracellular sugar levels and deal with potentially toxic phosphosugars cease growth and risk being outcompeted. Here, we identify the conserved haloacid dehalogenase (HAD)-like enzyme YigL as the previously hypothesized phosphatase for detoxification of phosphosugars and reveal that its synthesis is activated by an Hfq-dependent small RNA in Salmonella typhimurium. We show that the glucose-6-P-responsive small RNA SgrS activates YigL synthesis in a translation-independent fashion by the selective stabilization of a decay intermediate of the dicistronic pldB-yigL messenger RNA (mRNA). Intriguingly, the major endoribonuclease RNase E, previously known to function together with small RNAs to degrade mRNA targets, is also essential for this process of mRNA activation. The exploitation of and targeted interference with regular RNA turnover described here may constitute a general route for small RNAs to rapidly activate both coding and noncoding genes.

Machine learning-aided engineering of hydrolases for PET depolymerization.

Plastic waste poses an ecological challenge1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling4. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste5, and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics11. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale.

Discovery of a Ni2+-dependent guanidine hydrolase in bacteria.

Nitrogen availability is a growth-limiting factor in many habitats1, and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogen-rich compound guanidine occurs widely in nature2-4, but its utilization is impeded by pronounced resonance stabilization5, and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni2+-dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni2+ instead of the typical Mn2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

(ADP-ribosyl)hydrolases: structure, function, and biology.

ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life. Its complexity derives from the varied range of different chemical linkages, including to several amino acid side chains as well as nucleic acids termini and bases, it can adopt. In this review, we provide an overview of the different families of (ADP-ribosyl)hydrolases. We discuss their molecular functions, physiological roles, and influence on human health and disease. Together, the accumulated data support the increasingly compelling view that (ADP-ribosyl)hydrolases are a vital element within ADP-ribosyl signaling pathways and they hold the potential for novel therapeutic approaches as well as a deeper understanding of ADP-ribosylation as a whole.

Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.

Phagocytes promptly resolve ingested targets to replenish lysosomes and maintain their responsiveness. The resolution process requires that degradative hydrolases, solute transporters, and proteins involved in lipid traffic are delivered and made active in phagolysosomes. It also involves extensive membrane remodeling. We report that cation channels that localize to phagolysosomes were essential for resolution. Specifically, the conductance of Na+ by two-pore channels (TPCs) and the presence of a Na+ gradient between the phagolysosome lumen and the cytosol were critical for the controlled release of membrane tension that permits deformation of the limiting phagolysosome membrane. In turn, membrane deformation was a necessary step to efficiently transport the cholesterol extracted from cellular targets, permeabilizing them to hydrolases. These results place TPCs as regulators of endomembrane remodeling events that precede target degradation in cases when the target is bound by a cholesterol-containing membrane. The findings may help to explain lipid metabolism dysfunction and autophagic flux impairment reported in TPC KO mice and establish stepwise regulation to the resolution process that begins with lysis of the target.

Misregulation of bromotyrosine compromises fertility in male Drosophila.

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes.

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.

Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2.

The regulation of membrane lipid composition is critical for cellular homeostasis. Cells are particularly sensitive to phospholipid saturation, with increased saturation causing membrane rigidification and lipotoxicity. How mammalian cells sense membrane lipid composition and reverse fatty acid (FA)-induced membrane rigidification is poorly understood. Here we systematically identify proteins that differ between mammalian cells fed saturated versus unsaturated FAs. The most differentially expressed proteins were two ER-resident polytopic membrane proteins: the E3 ubiquitin ligase RNF145 and the lipid hydrolase ADIPOR2. In unsaturated lipid membranes, RNF145 is stable, promoting its lipid-sensitive interaction, ubiquitination and degradation of ADIPOR2. When membranes become enriched in saturated FAs, RNF145 is rapidly auto-ubiquitinated and degraded, stabilising ADIPOR2, whose hydrolase activity restores lipid homeostasis and prevents lipotoxicity. We therefore identify RNF145 as a FA-responsive ubiquitin ligase which, together with ADIPOR2, defines an autoregulatory pathway that controls cellular membrane lipid homeostasis and prevents acute lipotoxic stress.

A lipid hydrolase and a ubiquitin ligase play hide-and-seek in the ER membrane.

Complex metabolic diseases such as diabetes and non-alcoholic fatty liver disease have been associated with aberrant lipid metabolism and lipotoxicity. To maintain lipid homeostasis and escape lipotoxicity, cells deploy a plethora of mechanisms, the most fascinating of which relying on a sense-and-response circuit. New work by Volkmar et al reveals an auto-regulated pathway formed by a lipid hydrolase and a lipid-sensitive E3 ubiquitin ligase playing hide-and-seek to warrant membrane function in stressed cells.

Unconventional post-translational modifications in immunological signaling.

The activity of a cell is governed by the signals it receives from the extracellular milieu, which are 'translated' into the appropriate biological output, such as activation, survival, proliferation, migration or differentiation. Signaling pathways are responsible for converting environmental cues into discrete intracellular events. The alteration of existing proteins by post-translational modification (PTM) is a key feature of signal-transduction pathways that allows the modulation of protein function. Research into PTMs has long been dominated by the investigation of protein phosphorylation; other PTMs, such as methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, have received comparatively little attention. This Review aims to present an overview of these PTMs, with an emphasis on their role in cells of the immune system.