Targeting integral membrane proteins in quantitative proteomics for medical applications and biotechnological issues by Corynebacterium glutamicum\textit {Corynebacterium glutamicum} and Escherichia coli\textit {Escherichia coli}

Es wurden bestehende Methoden für die Shotgun-Proteomics genutzt und in Bezug auf die herausfordernde Analyse von Membranproteinen optimiert. Diese Methodenentwicklung ermöglichte es, umfassende Proteomstudien zu biotechnologisch relevanten Fragestellungen zu bearbeiten. Der Schwerpunkt dieser Arbeit befasst sich mit Quantitativer Membranproteomanalytik. Dazu wurde ein Membranprotein-Anreicherungsverfahren, das Biphasische System, etabliert und gezeigt, dass sich dieses auch zur relativen Quantifizierung eignet. Hierbei erfolgten die globale Analyse des L-Lysin-Produzenten im Vergleich zum Wildtyp von $\textit {C. glutamicum}$ und die Anpassung von $\it {C. glutamicum}$ bei Salzstress. Darüber hinaus wurde die medizinische Grundlagenforschung vorangetrieben, indem insbesondere die Erforschung der neuartigen Antibiotikagruppe der kleinen Metall-konjugierten Peptide erfolgte. Die in diesen globalen Studien weiterführenden Fragen wurden zusätzlich mit biochemischen und biophysikalischen Methoden geklärt

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis

In amanner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown totarget proteins for degradation via the proteasome inmycobacteria. However, not all actinobacteriapossessing the Pup protein also contain a proteasome. In this study, we set out to studypupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum.A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as theparent strain in standard glucose minimal medium, indicating that pupylation is dispensableunder these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidinetag in the pup mutant and Ni2+-chelate affinity chromatography, pupylated proteinswere isolated. Multidimensional protein identification technology (MudPIT) andMALDI-TOFMS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved inmetabolism or translation. Our results define the first pupylome of an actinobacterial specieslacking a proteasome, confirming that other fates besides proteasomal degradation are possiblefor pupylated proteins

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.14 page(s

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

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 $\textit {Sordaria macrospora}$. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated $\it mik1$ 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

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

<div><p>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 <i>Sordaria macrospora</i>. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated <i>mik1</i> 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.</p></div

PRO40 is required for correct signaling via the CWI pathway.

<p>(A) Time course of MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three to six days, and phosphorylated MAK1 was detected in a Western blot using an anti-phospho-p44/42 antibody. The signal for tubulin was used as internal standard. Representative immunoblots of two to four independent experiments with three technical replicates are shown. (B) Stress-induced MAK1 phosphorylation in <i>pro40</i> deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three days and subjected to 0.01% H₂O₂ for 0, 15, 30, and 45 minutes prior to harvesting. Phosphorylated MAK1 was detected using an anti-phospho-p44/42 antibody, and the signal for tubulin was used as internal standard. Representative immunoblots of three independent experiments with three technical replicates are shown. (C) Model of the scaffolding function of PRO40 for the CWI pathway. Details are discussed in the text.</p

The Δmek1/pro40 double mutants shares phenotypic characteristics with Δmek1 and Δpro40.

<p>(A) Sexual development was assayed after 7 days of growth on BMM slides. Δpro40 and the Δmek1/pro40 double mutant generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) Δpro40 and the Δmek1/pro40 double mutant are unable to undergo hyphal fusion, although hyphae often grow in close contact (white arrowheads). Scale bar, 10 µm. (C) Localization of GFP-tagged MIK1, MEK1, and MAK1 in vegetative hyphae of the pro40 mutant and Δpro40. Scale bar, 10 µm. (D) Localization of GFP-tagged MIK1, MEK1, and MAK1 in three days old protoperithecia of the wildtype, the pro40 mutant, and the <i>pro40</i> deletion strain Δpro40. Scale bar, 20 µm.</p