Liver steatosis is associated with the development of insulin resistance and the pathogenesis of type 2 diabetes. We tested the hypothesis that protein signals originating from steatotic hepatocytes communicate with other cells to modulate metabolic phenotypes. We show that the secreted factors from steatotic hepatocytes induce pro-inflammatory signaling and insulin resistance in cultured cells. Next, we identified 168 hepatokines, of which 32 were differentially secreted in steatotic versus non-steatotic hepatocytes. Targeted analysis showed that fetuin B was increased in humans with liver steatosis and patients with type 2 diabetes. Fetuin B impaired insulin action in myotubes and hepatocytes and caused glucose intolerance in mice. Silencing of fetuin B in obese mice improved glucose tolerance. We conclude that the protein secretory profile of hepatocytes is altered with steatosis and is linked to inflammation and insulin resistance. Therefore, preventing steatosis may limit the development of dysregulated glucose metabolism in settings of overnutrition.

Relative label-free quantification (LFQ) of shotgun proteomics data using precursor (MS1) signal intensities is one of the most commonly used applications to comprehensively and globally quantify proteins across biological samples and conditions. Due to the popularity of this technique, several software packages, such as the popular software suite MaxQuant, have been developed to extract, analyze, and compare spectral features and to report quantitative information of peptides, proteins, and even post-translationally modified sites. However, there is still a lack of accessible tools for the interpretation and downstream statistical analysis of these complex data sets, in particular for researchers and biologists with no or only limited experience in proteomics, bioinformatics, and statistics. We have therefore created LFQ-Analyst, which is an easy-to-use, interactive web application developed to perform differential expression analysis with "one click" and to visualize label-free quantitative proteomic data sets preprocessed with MaxQuant. LFQ-Analyst provides a wealth of user-analytic features and offers numerous publication-quality result graphics to facilitate statistical and exploratory analysis of label-free quantitative data sets. LFQ-Analyst, including an in-depth user manual, is freely available at https:// bioinformatics.erc.monash.edu/apps/LFQ-Analyst.

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

Non-ribosomal peptide biosynthesis produces highly diverse natural products through a complex cascade of enzymatic reactions that together function with high selectivity to produce bioactive peptides. The modification of non-ribosomal peptide synthetase (NRPS)-bound amino acids can introduce significant structural diversity into these peptides and has exciting potential for biosynthetic redesign. However, the control mechanisms ensuring selective modification of specific residues during NRPS biosynthesis have previously been unclear. Here, we have characterised the incorporation of the non-proteinogenic amino acid 3-chloro-b-hydroxytyrosine during glycopeptide antibiotic (GPA) biosynthesis. Our results demonstrate that the modification of this residue by trans-acting enzymes is controlled by the selectivity of the upstream condensation domain responsible for peptide synthesis. A proofreading thioesterase works together with this process to ensure that effective peptide biosynthesis proceeds even when the selectivity of key amino acid activation domains within the NRPS is low. Furthermore, the exchange of condensation domains with altered amino acid specificities allows the modification of such residues within NRPS biosynthesis to be controlled, which will doubtless prove important for reengineering of these assembly lines. Taken together, our results indicate the importance of the complex interplay of NRPS domains and trans-acting enzymes to ensure effective GPA biosynthesis, and in doing so reveals a process that is mechanistically comparable to the hydrolytic proofreading function of tRNA synthetases in ribosomal protein synthesis.Peptide synthesis for tripeptide 3T-CoA, tetrapeptide 4T-CoA and pentapeptide 5T-CoA CoAs was performed manually by 9470 | Chem. Sci., 2019, 10, 9466-9482This journal is