Maarten Altelaar

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division. DOI: http://dx.doi.org/10.7554/eLife.07118.001

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush.

Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell–Specific Proteases

Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue–type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9+ EV, degranulation was accompanied by the release of CD63+ EV, which correlated with release of the soluble mediator β-hexosaminidase. This CD63+ EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9+ EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63+ EV from connective tissue–type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.

Cultivation of Podospora anserina on soybean hulls results in an efficient enzyme cocktail for plant biomass hydrolysis

The coprophilic ascomycete fungus Podospora anserina was cultivated on three different plant biomasses, i.e. cotton seed hulls (CSH), soybean hulls (SBH) and acid-pretreated wheat straw (WS) for four days, and the potential of the produced enzyme mixtures was compared in the enzymatic saccharification of the corresponding lignocellulose feedstocks. The enzyme cocktail P. anserina produced after three days of growth on SBH showed superior capacity to release reducing sugars from all tested plant biomass feedstocks compared to the enzyme mixtures from CSH and WS cultures. Detailed proteomics analysis of the culture supernatants revealed that SBH contained the most diverse set of enzymes targeted on plant cell wall polymers and was particularly abundant in xylan, mannan and pectin acting enzymes. The importance of lytic polysaccharide monooxygenases (LPMOs) in plant biomass deconstruction was supported by identification of 20 out of 33 AA9 LPMOs in the SBH cultures. The results highlight the suitability of P. anserina as a source of plant cell wall degrading enzymes for biotechnological applications and the importance of selecting the most optimal substrate for the production of enzyme mixtures.

Proteomic and metabolomic analyses of vanishing white matter mouse astrocytes reveal deregulation of ER functions

Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation. We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.

The HAUS Complex Is a Key Regulator of Non-centrosomal Microtubule Organization during Neuronal Development

Summary Neuron morphology and function are highly dependent on proper organization of the cytoskeleton. In neurons, the centrosome is inactivated early in development, and acentrosomal microtubules are generated by mechanisms that are poorly understood. Here, we show that neuronal migration, development, and polarization depend on the multi-subunit protein HAUS/augmin complex, previously described to be required for mitotic spindle assembly in dividing cells. The HAUS complex is essential for neuronal microtubule organization by ensuring uniform microtubule polarity in axons and regulation of microtubule density in dendrites. Using live-cell imaging and high-resolution microscopy, we found that distinct HAUS clusters are distributed throughout neurons and colocalize with γ-TuRC, suggesting local microtubule nucleation events. We propose that the HAUS complex locally regulates microtubule nucleation events to control proper neuronal development.

The physiology of Agaricus bisporus in semi-commercial compost cultivation appears to be highly conserved among unrelated isolates

The white button mushroom Agaricus bisporus is one of the most widely produced edible fungus with a great economical value. Its commercial cultivation process is often performed on wheat straw and animal manure based compost that mainly contains lignocellulosic material as a source of carbon and nutrients for the mushroom production. As a large portion of compost carbohydrates are left unused in the current mushroom cultivation process, the aim of this work was to study wild-type A. bisporus strains for their potential to convert the components that are poorly utilized by the commercial strain A15. We therefore focused our analysis on the stages where the fungus is producing fruiting bodies. Growth profiling was used to identify A. bisporus strains with different abilities to use plant biomass derived polysaccharides, as well as to transport and metabolize the corresponding monomeric sugars. Six wild-type isolates with diverse growth profiles were compared for mushroom production to A15 strain in semi-commercial cultivation conditions. Transcriptome and proteome analyses of the three most interesting wild-type strains and A15 indicated that the unrelated A. bisporus strains degrade and convert plant biomass polymers in a highly similar manner. This was also supported by the chemical content of the compost during the mushroom production process. Our study therefore reveals a highly conserved physiology for unrelated strains of this species during growth in compost.

Abstract B9: A phosphoproteomic portrait of triple - negative breast cancer: functional taxonomy

BACKGROUND: Triple-negative breast cancer (TNBC) is a breast cancer subtype that is not defined by targetable molecular markers. Since all the aberrations that exist and ultimately contribute to the tumor phenotype converge, from a functional point of view, in the final status of the phosphorylation of the proteome in a given moment, we sought to interrogate the phosphoproteome with two aims: 1) establish a taxonomy of TNBC based on measurable markers that predict clinical course; 2) reduce the phosphoproteome that characterizes the bad- from good-prognosis cases to its targetable, driving kinases, in order to define rational therapeutic approaches in TNBC. We chose mass spectrometry as a discovery approach, because of its coverage, sensitivity, dynamic range and specificity. Here we report the part of phosphoproteomic analysis used for establishing a new taxonomy of TNBC. METHODS: We performed quantitative phosphoproteomics from a discovery set of 34 frozen tumor samples divided in two sets paired by classic prognosis factors: 1, with 13 patients relapsed in less than 3 years; 2, 21 patients, no relapse in 10 years follow up. Raw data were processed with Maxquant and cases clustered using a supervised hierarchical approach. Kinases (predicted with linear domain consensus analysis tools using a PHOSIDA) and phospho-sites discriminating the set 1 from 2 were validated by immunohistochemistry in 113 consecutive TNBC cases, spotted in TMAs, with 14 years follow up. RESULTS: Overall, 15000 unique phosphopeptides were identified. Supervised clustering of subset 1 vs. 2 showed that 161 and 541 peptides were significantly more phosphorylated in the subset 1 and 2, respectively (FDR Kinase validation was performed directing an antibody against the active kinase form, if available; if not, against the total levels. The staining of kinases and the candidate phosphosites was analyzed with an Ariol, scored with a continue value from 0 to 3, and divided in quartiles for each staining probe. So far, 12 probes have been quantified. The prognosis of patients with upper quartile staining vs. the remainder was analyzed with the log-rank test and cox proportionate hazards model for each probe. Five of them showed statistically significant association with relapse (pSTAT3Tyr705, 6.1 vs. 10.1 years, p = 0.024; CYCB1, 7 vs. 10.3 years, p = 0.027; CDK6, 7 vs. 10.3 years, p = 0.013; pp70S6KThr389, 7.2 vs. 10 years, p = 0.042 and PRKCEpsilon, 7.1 vs. 10.2 years, p = 0.021). A combined variable integrating upper-quartile staining from either of those 5 probes occurring in 67.4% patients, identified almost all patients that experienced relapse: 48.3% of them relapsed (median time 7.6 years) vs. the reminder 32.6% of which only 13.8% relapsed (median time 11.9 years), log rank p = 0.001 INTERPRETATION: High-throughput mass-spectrometry is a powerful tool for generating a disease taxonomy, and can be translated to routine techniques like IHC. The signature constituted by pSTAT3Tyr705, CYCB1, CDK6, pp70S6K and PRKCEpsilon constitutes the most parsimonious signature to date able to detect all the early TNBC patients that relapse in the long term. Citation Format: Ivana Zagorac, Tamara Mondejar Tevar, Jesus Sanchez Ruiz, Albert Heck, Maarten Altelaar, Renske Penning, Harm Post, Gonzalo Gomez Lopez, David Pisano, Javier Munoz Peralta, Javier Munoz Peralta, Manuel Morente, Luis Manso, Miguel Quintela-Fandino. A phosphoproteomic portrait of triple - negative breast cancer: functional taxonomy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B9.

In-depth proteomics analysis of human breast milk-derived extracellular vesicles to reveal their origin and targets in the infant's developing immune system

Introduction: Besides providing nutrition, breast milk delivers important signals that stimulate the infants developing immune system. It has been postulated that extracellular vesicles (EV) in milk support the instruction and/or development of neonatal immunity. However, little is known about the composition of milk-derived EV, partly due to the difficulty to purify EV from other components in milk. Methods: In this study, an extensive LC-MS/MS proteomic analysis was performed, whereby EV were isolated from breast milk of 7 individual donors using our recently established optimized density gradient-based isolation protocol [1]. High-density, non-floating complexes were included to compare the contents of EV to other macromolecular structures in milk. A comprehensive protein network was composed tracing the possible cellular origins of milk-derived EV and the potential targets in the gut. Results: An average of 579 proteins was identified in EV, compared to 205 proteins in the non-floating fraction. Interestingly, EV associated proteins like ANXA5 and Flotillin were exclusively identified in EV, while CD9, CD63 and CD81 were also present in non-floating protein complexes. Additionally, MHC-II was identified in the EV fraction only, suggesting that antigenic epitopes may be delivered via EV released from antigen-presenting cells. Besides MHC-I, the mammary epithelial cell marker beta-1,4-galactosyltransferase (lactose subunit) was identified in the EV fraction only, demonstrating EV of epithelial origin. Furthermore, several adhesion molecules (ICAM-1, CEACAM-1) were associated to EV which could allow EV binding to gut epithelial cells and gut resident immune cells. Summary/conclusion: In-depth proteomic analysis and compilation of an extensive network of EV proteins involved in immunity demonstrates that milk-derived EV originate from multiple cellular sources and have the ability to target various cell types in the gut.ISEV 2015 is organized by The Local Organizing Committee: Kenneth Witwer (Chair, Baltimore), Shilpa Buch (Omaha), Prasun Datta (Philadelphia), Dolores Di Vizio (Los Angeles), Uta Erdbrügger (Charlottesville), Steven Jay (College Park), Dimitrios Kapogiannis (Baltimore), Leonid Margolis (Bethesda) & Susmita Sahoo (New York) Together with the Executive ISEV Board (2014 – 2016) President: Jan Lötvall Secretary General: Clotilde Théry Treasurer: Fred Hochberg Executive Chair Science / Meetings: Marca Wauben Executive Chair Education: Yong Song Gho Executive Chair Communication: Andrew Hill Members at Large: Peter Quesenberry, Kenneth Witwer, Susmita Sahoo, Dolores Di Vizio, Chris Gardiner, Edit Buzas, Hidetoshi Tahara, Suresh Mathivanan, Igor Kurochkin