Sequence-Selective Covalent CaaX-Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3†K Signaling.

Oncogenic Ras proteins are implicated in the most common life-threatening cancers. Despite intense research over the past two decades, the progress towards small-molecule inhibitors has been limited. One reason for this failure is that Ras proteins interact with their effectors only via protein-protein interactions, which are notoriously difficult to address with small organic molecules. Herein we describe an alternative strategy, which prevents farnesylation and subsequent membrane insertion, a prerequisite for the activation of Ras proteins. Our approach is based on sequence-selective supramolecular receptors which bind to the C-terminal farnesyl transferase recognition unit of Ras and Rheb proteins and covalently modify the essential cysteine in the so-called CaaX-box.

Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, l-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In addition, treatment with AngII led to LCP1 redistribution to the cell margins, membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in podocytes.-Schenk, L. K., Möller-Kerutt, A., Klosowski, R., Wolters, D., Schaffner-Reckinger, E., Weide, T., Pavenstädt, H., Vollenbröker, B. Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Strategies in relative and absolute quantitative mass spectrometry based proteomics.

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting.

Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns.

In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaA-i2, splice in trans, and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatography-mass spectrometry (LC-MS), size exclusion chromatography, and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein apparatus participating in psaA mRNA splicing. This supercomplex contains two subcomplexes (I and II) that are responsible for trans-splicing of either psaA-i1 or psaA-i2. We further demonstrate that both subcomplexes are associated with intron RNA, which is a prerequisite for the correct assembly of subcomplex I. This study contributes further to our view of how the eukaryotic nuclear spliceosome evolved after bacterial endosymbiosis through fragmentation of self-splicing group II introns into a dynamic, protein-rich RNP machinery.

Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

UNLABELLED: The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. IMPORTANCE: The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood. The first fungal STRIPAK was described in Sordaria macrospora, which is a well-established model organism used to study the formation of fungal fruiting bodies, three-dimensional organ-like structures. We analyzed STRIPAK subunit PP2Ac1, catalytic subunit 1 of protein phosphatase PP2A, to study the importance of the catalytic activity of this protein during sexual development. The results of our yeast two-hybrid analysis and tandem affinity purification, followed by mass spectrometry, indicate that PP2Ac1 activity connects STRIPAK with other signaling pathways and thus forms a large interconnected signaling network.

Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2.

Mutations in the gene coding for the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) are the leading cause of genetically inherited Parkinson's disease (PD). Two of the common found mutations are the R1441C and G2019S. In this study we identified protein phosphatase 2A (PP2A) as an interacting partner of LRRK2. We were able to demonstrate that the Ras of complex protein (ROC) domain is sufficient to interact with the three subunits of PP2A in human neuroblastoma SH-SY5Y cells and in HeLa cells. The alpha subunit of PP2A is interacting with LRRK2 in the perinuclear region of HeLa cells. Silencing the catalytic subunit of PP2A by shRNA aggravated cellular degeneration induced by the pathogenic R1441C-LRRK2 mutant expressed in neuroblastoma SH-SY5Y cells. A similar enhancement of apoptotic nuclei was observed by downregulation of the catalytic subunit of PP2A in cultured cortical cells derived from neurons overexpressing the pathogenic mutant G2019S-LRRK2. Conversely, pharmacological activation of PP2A by sodium selenate showed a partial neuroprotection from R1441C-LRRK2-induced cellular degeneration. All these data suggest that PP2A is a new interacting partner of LRRK2 and reveal the importance of PP2A as a potential therapeutic target in PD.

A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii.

Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity.

Biochemical Large-Scale Interaction Analysis of Murine Olfactory Receptors and Associated Signaling Proteins with Post-Synaptic Density 95, Drosophila Discs Large, Zona-Occludens 1 (PDZ) Domains.

G protein-coupled receptors (GPCRs) constitute the largest family among mammalian membrane proteins and are capable of initiating numerous essential signaling cascades. Various GPCR-mediated pathways are organized into protein microdomains that can be orchestrated and regulated through scaffolding proteins, such as PSD-95/discs-large/ZO1 (PDZ) domain proteins. However, detailed binding characteristics of PDZ-GPCR interactions remain elusive because these interactions seem to be more complex than previously thought. To address this issue, we analyzed binding modalities using our established model system. This system includes the 13 individual PDZ domains of the multiple PDZ domain protein 1 (MUPP1; the largest PDZ protein), a broad range of murine olfactory receptors (a multifaceted gene cluster within the family of GPCRs), and associated olfactory signaling proteins. These proteins were analyzed in a large-scale peptide microarray approach and continuative interaction studies. As a result, we demonstrate that canonical binding motifs were not overrepresented among the interaction partners of MUPP1. Furthermore, C-terminal phosphorylation and distinct amino acid replacements abolished PDZ binding promiscuity. In addition to the described in vitro experiments, we identified new interaction partners within the murine olfactory epithelium using pull-down-based interactomics and could verify the partners through co-immunoprecipitation. In summary, the present study provides important insight into the complexity of the binding characteristics of PDZ-GPCR interactions based on olfactory signaling proteins, which could identify novel clinical targets for GPCR-associated diseases in the future.

Proteinase K improves quantitative acylation studies.

Acetylation is a common PTM of proteins but is still challenging to analyze. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestion conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites.

A fungal sarcolemmal membrane-associated protein (SLMAP) homolog plays a fundamental role in development and localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria.

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.

Patterns of Proteins Removed with High-Flux Membranes on High-Volume Hemodiafiltration Detected with a MultiDimensional LC-MS/MS Strategy.

Background: Aim of this prospective crossover study was to identify the nature of the middle-molecular weight solutes removed during high-volume post-dilution HDF. Methods: The efficiency in removing small molecules, protein-bound and middle-molecular proteins was evaluated in 16 chronic dialysis patients on post-dilution HDF with two high-flux dialyzer membranes (Amembris and Polyamix). Multidimensional Protein Identification Technology (MudPIT) was employed to identify middle-molecular weight solutes in spent dialysate. Results: Efficiency of post-dilution HDF in removing solutes of different MW was high with both membranes, but higher with Amembris than with Polyamix. With MudPIT analysis, 277 proteins were identified in the dialysate fluids. Although the protein-removal pattern was similar among patients and tested membranes, the total and protein-specific peptide spectral count (mass spectrometric quantitation criteria) of most proteins were higher using the Amembris membrane. Conclusions: The MudPIT approach showed to be a powerful tool to identify a broad molecular weight spectrum of proteins removed with post-dilution HDF. Short Summary: Aim of this prospective crossover study was to analyze the hydraulic properties of two high-flux dialyzer membranes (Amembris and Polyamix) during high-volume, post-dilution HDF and to evaluate the influence of these properties on the removal of proteins and peptides using an in-depth analysis of the spent dialysate. For this analysis, a liquid chromatography tandem mass spectrometry approach called MudPIT (Multidimensional Protein Identification Technology) was used to identify the middle molecular weight solutes present in the spent dialysate of patients. The capability of post-dilution HDF in removing solutes of different MW was very high with both dialyzers, but higher with the Amembris membrane. The proteomic MudPIT approach showed to be a powerful tool to identify a wide molecular spectrum of proteins removed from blood during post-dilution HDF. These results may contribute to address research toward a better knowledge of uremic toxins and the balance between the intended and unintended removal of undesired and beneficial proteins next to identification of new target proteins as potential candidates for uremic toxicity. © 2014 S. Karger AG, Basel.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis.

In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.

The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway.

The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.

Coxsackievirus B3 modulates cardiac ion channels.

Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.

Proteome analysis reveals antiangiogenic environments in chronic wounds of diabetes mellitus type 2 patients.

In contrast to normal healing wounds, chronic wounds commonly show disturbances in proteins regulating wound healing processes, particularly those involved in cell proliferation and protein degradation. Multidimensional protein identification technology MS/MS was conducted to investigate and compare the protein composition of chronic diabetic foot exudates to exudates from split-skin donor sites of burn victims otherwise healthy. Spectral counting revealed 188 proteins differentially expressed (more than twofold and p-value <0.05) in chronic wounds. Most were involved in biological processes including inflammation, angiogenesis, and cell mortality. Increased expression of the inflammatory response stimulating S100 proteins, predominantly S100A8 and S100A9 (almost tenfold), was identified. Matrix metalloproteinases (MMPs) MMP1, MMP2, and MMP8 were identified to be elevated in chronic wounds with significant impact on collagen degradation and tissue destruction. Further, proteins with antiangiogenic properties were found at higher expression levels in chronic wounds. Reduced angiogenesis leads to drastic shortage in nutrition supply and causes increased cell death, demonstrated by Annexin A5 exclusively found in chronic wound exudates. However, excessive nucleic and cytosolic material infers cell death occurring not only by apoptosis but also by necrosis. In conclusion, mass spectrometric investigation of exudates from chronic wounds demonstrated dramatic impairment in wound repair with excessive inflammation, antiangiogenic environment, and accelerated cell death.

Identification of a chloroplast ribonucleoprotein complex containing trans-splicing factors, intron RNA, and novel components.

Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns.

Physiological adaptation of the Rhodococcus jostii RHA1 membrane proteome to steroids as growth substrates.

Rhodococcus jostii RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen Mycobacterium tuberculosis , are discussed.

A molecular mechanism for direct sirtuin activation by resveratrol.

Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuin's polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair.