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There is growing interest in reducing friction in lubricated machine components to thereby increase the energy efficiency of machines. One important way to minimise friction is to employ friction modifier additives to reduce friction in thin film boundary lubrication conditions. There are currently three main types of friction modifier additive, organic friction modifiers, oil soluble organomolybdenum friction modifiers and functionalised polymers. In common practice, a single such additive is generally employed in a formulated lubricant, but it is of interest to explore whether combinations of two friction modifier additives may prove beneficial. In this study, the performance of eight commercial friction modifier additives spanning all three main types was first measured in three quite different friction tests. The aim was to identify the contact conditions under which each additive was most effective. Additive solutions in both a base oil and a formulated engine oil were investigated. In general, functionalised polymers were most beneficial in sliding–rolling contacts, while oil soluble organomolybdenum friction modifiers worked best in severe, reciprocating sliding conditions. However, all friction modifier additive response was strongly affected by the other additives present in formulated engine oils. The friction performance of combinations of friction modifier additives was then explored. When two different friction modifiers additives were combined in solution, several possible outcomes were observed. The most common was for one of the additives to predominate, to give friction that was characteristic of that additive alone, while in some cases friction lay between the values produced by either additive on its own. In a few cases the additives behaved antagonistically so that the combination gave higher friction than either additive by itself. In a few cases true synergy was observed, where a combination of two additives produced lower friction in a given test that either individual component at the same overall concentration. Another, and possibly more important synergy could also occur, however, when a pair of FMs worked more effectively than either individual additive over the range of test conditions present in different friction tests. This study suggests that optimal combinations of FMs may provide a means of reducing boundary friction and thus increasing the efficiency of machines, especially if the latter contain a range of lubricated machine components that operate with different types of tribological contacts.

The ATG12~ATG5 conjugate promotes the transfer of the ubiquitin-like protein LC3 to phosphatidylethanolamine (PE), a modification required for autophagosome formation. Structural and biochemical analyses reveal the determinants for ATG12~ATG5 binding to ATG16 and the E3 ligase ATG3, and indicate how the conjugate stimulates PE–LC3 formation. The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase–like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous surface patch. Mutagenesis data support the importance of both the interface between ATG12 and ATG5 and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

[This corrects the article DOI: 10.1371/journal.pone.0272826.].

SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.

SUMOylation is a dynamic protein post-translational modification that regulates many eukaryotic proteins. Now a methodology using commercially available monoclonal antibodies coupled to MS analysis leads to the enrichment and identification of endogenous targets for SUMO1 and for SUMO2/3 in HeLa cells and mouse liver. This protocol can be adapted for other tissues and organs. SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.

This study aimed to evaluate the prognostic value and biological function of small ubiquitin‐like modifier 2 (SUMO2) in hepatocellular carcinoma (HCC). SUMO2 expression in HCC tissues was markedly higher than that in normal liver tissues, and patients with high SUMO2 expression had significantly shorter median overall survival than those with low SUMO2 expression. Furthermore, SUMO2 expression was closely correlated with lymph node metastasis and vascular invasion and was a predictor of poor prognosis. The knockdown of SUMO2 in two HCC cell lines (SMMC‐7721 and Bel‐7404) dramatically suppressed their proliferation, migration and invasion. Western blot analysis showed that the downregulation of SUMO2 significantly reduced the expression of Ki‐67, matrix metalloproteinase‐9 (MMP‐9) and vascular endothelial growth factor (VEGF) in SMMC‐7721 and Bel‐7404 cells. Similarly, quantitative reverse transcription–PCR revealed consistently decreased expression of MMP‐9 and VEGF. Our data suggest that SUMO2 promotes proliferation, migration and invasion of HCC cells via mechanisms involving MMP‐9 and VEGF. Therefore, SUMO2 may be a prognostic factor and a promising therapeutic target for patients with HCC. We observed that the overexpression of small ubiquitin‐like modifier 2 (SUMO2) in hepatocellular carcinoma (HCC) tissues is associated with poor prognosis in patients with HCC. Silencing of SUMO2 in HCC cell lines reduced the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase‐9 (MMP‐9). SUMO2 promoted the tumorigenic phenotypes of HCC cells through mechanisms involving MMP‐9 and VEGF. Therefore, SUMO2 may serve as a prognostic marker and a promising therapeutic target for patients with HCC.

DNA damage repair genes are modifiers of disease onset in Huntington’s disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Post-translational SUMO modification is a widespread mechanism for regulating protein function within cells. In humans, SUMO-conjugated proteins are partially regulated by the deconjugating activity of six SENP family members. The proteolytic activity of these enzymes resides within a conserved catalytic domain that exhibits specificity for the two primary SUMO isoforms: SUMO1 and SUMO2/3. SENP5, along with SENP3, are nucleolar proteins involved in ribosome biogenesis and preferentially target SUMO2/3 isoforms. Here, we present the crystal structures of human SENP5 in complex with both SUMO1 and SUMO2 isoforms. These structures reveal a minimal complex interface and elucidate the molecular basis for SENP5’s preference for the SUMO2 isoform. This preference can be attributed to a basic patch surrounding SENP5 Arg624 at the interface. Swapping mutagenesis and structural analysis demonstrate that Arg624 is favorably oriented to interact with Asp63 in SUMO2/3, while its interaction with the equivalent Glu67 in SUMO1 is less favorable. These results suggest that subtle structural differences within SUMO isoforms can significantly influence their deconjugation by SENP enzymes, opening new avenues for exploring the regulation of SUMOylation in various cellular processes and for developing therapeutic agents targeting SUMOylation pathways. SUMO modification regulates protein function, with SENP enzymes controlling SUMO removal. Here, the authors present crystal structures of SENP5 bound to SUMO1 and SUMO2, revealing how structural features drive its preference for SUMO2 and offering insights into SUMOylation regulation.