The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Proteasome Inhibitors: Harnessing Proteostasis to Combat Disease.

The proteasome is the central component of the main cellular protein degradation pathway. During the past four decades, the critical function of the proteasome in numerous physiological processes has been revealed, and proteasome activity has been linked to various human diseases. The proteasome prevents the accumulation of misfolded proteins, controls the cell cycle, and regulates the immune response, to name a few important roles for this macromolecular "machine." As a therapeutic target, proteasome inhibitors have been approved for the treatment of multiple myeloma and mantle cell lymphoma. However, inability to sufficiently inhibit proteasome activity at tolerated doses has hampered efforts to expand the scope of proteasome inhibitor-based therapies. With emerging new modalities in myeloma, it might seem challenging to develop additional proteasome-based therapies. However, the constant development of new applications for proteasome inhibitors and deeper insights into the intricacies of protein homeostasis suggest that proteasome inhibitors might have novel therapeutic applications. Herein, we summarize the latest advances in proteasome inhibitor development and discuss the future of proteasome inhibitors and other proteasome-based therapies in combating human diseases.

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.

The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site.

Community-scale models of microbiomes: Articulating metabolic modelling and metagenome sequencing.

Building models is essential for understanding the functions and dynamics of microbial communities. Metabolic models built on genome-scale metabolic network reconstructions (GENREs) are especially relevant as a means to decipher the complex interactions occurring among species. Model reconstruction increasingly relies on metagenomics, which permits direct characterisation of naturally occurring communities that may contain organisms that cannot be isolated or cultured. In this review, we provide an overview of the field of metabolic modelling and its increasing reliance on and synergy with metagenomics and bioinformatics. We survey the means of assigning functions and reconstructing metabolic networks from (meta-)genomes, and present the variety and mathematical fundamentals of metabolic models that foster the understanding of microbial dynamics. We emphasise the characterisation of interactions and the scaling of model construction to large communities, two important bottlenecks in the applicability of these models. We give an overview of the current state of the art in metagenome sequencing and bioinformatics analysis, focusing on the reconstruction of genomes in microbial communities. Metagenomics benefits tremendously from third-generation sequencing, and we discuss the opportunities of long-read sequencing, strain-level characterisation and eukaryotic metagenomics. We aim at providing algorithmic and mathematical support, together with tool and application resources, that permit bridging the gap between metagenomics and metabolic modelling.

Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli.

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS. A further ~10% of peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of atypical peptides. Our results suggest that generation of MHC class I•peptide complexes is more complex than previously recognized, with UPS-dependent and UPS-independent components; paradoxically, alternative protein degradation pathways also generate class I peptides when canonical pathways are impaired.

Comparative genomics of protoploid Saccharomycetaceae.

Our knowledge of yeast genomes remains largely dominated by the extensive studies on Saccharomyces cerevisiae and the consequences of its ancestral duplication, leaving the evolution of the entire class of hemiascomycetes only partly explored. We concentrate here on five species of Saccharomycetaceae, a large subdivision of hemiascomycetes, that we call "protoploid" because they diverged from the S. cerevisiae lineage prior to its genome duplication. We determined the complete genome sequences of three of these species: Kluyveromyces (Lachancea) thermotolerans and Saccharomyces (Lachancea) kluyveri (two members of the newly described Lachancea clade), and Zygosaccharomyces rouxii. We included in our comparisons the previously available sequences of Kluyveromyces lactis and Ashbya (Eremothecium) gossypii. Despite their broad evolutionary range and significant individual variations in each lineage, the five protoploid Saccharomycetaceae share a core repertoire of approximately 3300 protein families and a high degree of conserved synteny. Synteny blocks were used to define gene orthology and to infer ancestors. Far from representing minimal genomes without redundancy, the five protoploid yeasts contain numerous copies of paralogous genes, either dispersed or in tandem arrays, that, altogether, constitute a third of each genome. Ancient, conserved paralogs as well as novel, lineage-specific paralogs were identified.

A community standard format for the representation of protein affinity reagents.

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.

Génolevures: protein families and synteny among complete hemiascomycetous yeast proteomes and genomes.

The Génolevures online database (http://cbi.labri.fr/Genolevures/ and http://genolevures.org/) provides exploratory tools and curated data sets relative to nine complete and seven partial genome sequences determined and manually annotated by the Génolevures Consortium, to facilitate comparative genomic studies of Hemiascomycete yeasts. The 2008 update to the Génolevures database provides four new genomes in complete (subtelomere to subtelomere) chromosome sequences, 50,000 protein-coding and tRNA genes, and in silico analyses for each gene element. A key element is a novel classification of conserved multi-species protein families and their use in detecting synteny, gene fusions and other aspects of genome remodeling in evolution. Our purpose is to release high-quality curated data from complete genomes, with a focus on the relations between genes, genomes and proteins.

A differential cell capture assay for evaluating antibody interactions with cell surface targets.

Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies' concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.

Family relationships: should consensus reign?--consensus clustering for protein families.

MOTIVATION: Reliable identification of protein families is key to phylogenetic analysis, functional annotation and the exploration of protein function diversity in a given phylogenetic branch. As more and more complete genomes are sequenced, there is a need for powerful and reliable algorithms facilitating protein families construction. RESULTS: We have formulated the problem of protein families construction as an instance of consensus clustering, for which we designed a novel algorithm that is computationally efficient in practice and produces high quality results. Our algorithm uses an election method to construct consensus families from competing clustering computations. Our consensus clustering algorithm is tailored to serve the specific needs of comparative genomics projects. First, it provides a robust means to incorporate results from different and complementary clustering methods, thus avoiding the need for an a priori choice that may introduce computational bias in the results. Second, it is suited to large-scale projects due to the practical efficiency. And third, it produces high quality results where families tend to represent groupings by biological function. AVAILABILITY: This method has been used for Génolevures project to compute protein families of Hemiascomycetous yeasts. The data are available online at http://cbi.labri.fr/Genolevures/fam/

Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge.

Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser-like manner across a protein bridge that connects the inner and outer membranes.

How to decide which are the most pertinent overly-represented features during gene set enrichment analysis.

BACKGROUND: The search for enriched features has become widely used to characterize a set of genes or proteins. A key aspect of this technique is its ability to identify correlations amongst heterogeneous data such as Gene Ontology annotations, gene expression data and genome location of genes. Despite the rapid growth of available data, very little has been proposed in terms of formalization and optimization. Additionally, current methods mainly ignore the structure of the data which causes results redundancy. For example, when searching for enrichment in GO terms, genes can be annotated with multiple GO terms and should be propagated to the more general terms in the Gene Ontology. Consequently, the gene sets often overlap partially or totally, and this causes the reported enriched GO terms to be both numerous and redundant, hence, overwhelming the researcher with non-pertinent information. This situation is not unique, it arises whenever some hierarchical clustering is performed (e.g. based on the gene expression profiles), the extreme case being when genes that are neighbors on the chromosomes are considered. RESULTS: We present a generic framework to efficiently identify the most pertinent over-represented features in a set of genes. We propose a formal representation of gene sets based on the theory of partially ordered sets (posets), and give a formal definition of target set pertinence. Algorithms and compact representations of target sets are provided for the generation and the evaluation of the pertinent target sets. The relevance of our method is illustrated through the search for enriched GO annotations in the proteins involved in a multiprotein complex. The results obtained demonstrate the gain in terms of pertinence (up to 64% redundancy removed), space requirements (up to 73% less storage) and efficiency (up to 98% less comparisons). CONCLUSION: The generic framework presented in this article provides a formal approach to adequately represent available data and efficiently search for pertinent over-represented features in a set of genes or proteins. The formalism and the pertinence definition can be directly used by most of the methods and tools currently available for feature enrichment analysis.

Genome Sequence of the Yeast Clavispora lusitaniae Type Strain CBS 6936.

Clavispora lusitaniae, an environmental saprophytic yeast belonging to the CTG clade of Candida, can behave occasionally as an opportunistic pathogen in humans. We report here the genome sequence of the type strain CBS 6936. Comparison with sequences of strain ATCC 42720 indicates conservation of chromosomal structure but significant nucleotide divergence.

Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model.

Gram-negative bacteria have a double-membrane cellular envelope that enables them to colonize harsh environments and prevents the entry of many clinically available antibiotics. A main component of most outer membranes is lipopolysaccharide (LPS), a glycolipid containing several fatty acyl chains and up to hundreds of sugars that is synthesized in the cytoplasm. In the past two decades, the proteins that are responsible for transporting LPS across the cellular envelope and assembling it at the cell surface in Escherichia coli have been identified, but it remains unclear how they function. In this Review, we discuss recent advances in this area and present a model that explains how energy from the cytoplasm is used to power LPS transport across the cellular envelope to the cell surface.

Mimoza: web-based semantic zooming and navigation in metabolic networks.

BACKGROUND: The complexity of genome-scale metabolic models makes them quite difficult for human users to read, since they contain thousands of reactions that must be included for accurate computer simulation. Interestingly, hidden similarities between groups of reactions can be discovered, and generalized to reveal higher-level patterns. RESULTS: The web-based navigation system Mimoza allows a human expert to explore metabolic network models in a semantically zoomable manner: The most general view represents the compartments of the model; the next view shows the generalized versions of reactions and metabolites in each compartment; and the most detailed view represents the initial network with the generalization-based layout (where similar metabolites and reactions are placed next to each other). It allows a human expert to grasp the general structure of the network and analyze it in a top-down manner CONCLUSIONS: Mimoza can be installed standalone, or used on-line at http://mimoza.bordeaux.inria.fr/ , or installed in a Galaxy server for use in workflows. Mimoza views can be embedded in web pages, or downloaded as COMBINE archives.

Lipopolysaccharide transport to the cell surface: periplasmic transport and assembly into the outer membrane.

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane.

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.