ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis

Deep and fast label-free Dynamic Organellar Mapping

ABSTRACTThe Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we have drastically enhanced the performance of DOMs through data-independent acquisition (DIA) mass spectrometry (MS). DIA-DOMs achieve twice the depth of our previous workflow in the same MS runtime, and substantially improve profiling precision and reproducibility. We leveraged this gain to establish flexible map formats scaling from rapid analyses to ultra-deep coverage. Our fastest format takes only ∼2.5h/map and enables high-throughput experimental designs. Furthermore, we introduce DOM-QC, an open-source software tool for in-depth standardized analysis of DOMs and other profiling data. We then applied DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which revealed a subset of Golgi proteins that cycle through endosomes. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.</jats:p

Abstract LB-235: Therapeutic human papillomavirus vaccine design based on epitopes identified on the tumor cell surface by mass spectrometry

Abstract Detailed knowledge about T cell epitopes, which are bona fide presented on the surface of human papillomavirus (HPV)-transformed cells, is essential for rational design of therapeutic HPV vaccines. HPV affects the cellular antigen processing machinery, thus not every epitope derived from viral proteins is presented on human leukocyte antigen (HLA) molecules. Even the presented epitopes are displayed in low abundance. In this study, we developed a highly sensitive nano-UPLC-ESI-MS3 multiple-reaction monitoring mass spectrometry (MS) approach for direct detection of low-abundant epitopes on the cell surface. Several web-based prediction algorithms were used to predict prospective epitopes from the HPV16 E6 and E7 proteins. These candidate epitopes were tested for actual HLA binding in cellular binding assays. The presence of binding peptides on HPV16-transformed cells was analyzed by our MS technology. Immunogenicity of candidate peptides was assessed in vitro with short-term and long-term T cell line experiments, generated from peripheral blood mononuclear cells of healthy donors, and in vivo for a selected HLA-A2-restricted epitope in HLA-A2/DR-1 transgenic mice. To ensure &amp;gt;95% population coverage, prospective HPV epitopes were predicted for the HLA supertypes HLA-A1, A2, A3, A11, A24, B7, and B15. Close to 500 peptides were tested in competition-based binding assays and multiple novel binders were identified. MS epitope detection was first established for HLA-A2 epitopes, and is now extended to the other supertypes. In the immunogenicity assays, several peptides induced robust IFN-γ responses. In conclusion, several new HPV16 E6 and E7 epitopes were identified and validated in this study. They represent promising candidates for inclusion into a therapeutic HPV vaccine. Validated epitopes are the basis of rational therapeutic vaccine design and are also important for immunomonitoring purposes. Citation Format: Stephanie Hoppe, Renata Blatnik, Julia P. Schessner, Lisa Dressler, Alina Steinbach, Jan Winter, Martin Wuehl, Alexandra Klevenz, Hadeel Khallouf, Angelika B. Riemer. Therapeutic human papillomavirus vaccine design based on epitopes identified on the tumor cell surface by mass spectrometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-235. doi:10.1158/1538-7445.AM2015-LB-235</jats:p

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum

ABSTRACTER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and ESCRT proteins as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that ESCRT proteins directly function in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.</jats:p

AlphaPept: a modern and open framework for MS-based proteomics

Abstract In common with other omics technologies, mass spectrometry (MS)-based proteomics produces ever-increasing amounts of raw data, making efficient analysis a principal challenge. A plethora of different computational tools can process the MS data to derive peptide and protein identification and quantification. However, during the last years there has been dramatic progress in computer science, including collaboration tools that have transformed research and industry. To leverage these advances, we develop AlphaPept, a Python-based open-source framework for efficient processing of large high-resolution MS data sets. Numba for just-in-time compilation on CPU and GPU achieves hundred-fold speed improvements. AlphaPept uses the Python scientific stack of highly optimized packages, reducing the code base to domain-specific tasks while accessing the latest advances. We provide an easy on-ramp for community contributions through the concept of literate programming, implemented in Jupyter Notebooks. Large datasets can rapidly be processed as shown by the analysis of hundreds of proteomes in minutes per file, many-fold faster than acquisition. AlphaPept can be used to build automated processing pipelines with web-serving functionality and compatibility with downstream analysis tools. It provides easy access via one-click installation, a modular Python library for advanced users, and via an open GitHub repository for developers

Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells

AbstractDuring initiation of antiviral and antitumour T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on MHC class I. Cross-presentation relies on the unique ‘leakiness’ of endocytic compartments in DCs, whereby internalised proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell-type specific effectors of endocytic escape. We devised an escape assay suitable for genetic screening and identified a pore-forming protein, perforin-2, as a dedicated effector exclusive to cross-presenting cells. Perforin-2 is recruited to antigen-containing compartments, where it undergoes maturation, releasing its pore-forming domain.Mpeg1-/-mice fail to efficiently prime CD8+T cells to cell-associated antigens, revealing an important role of perforin-2 in cytosolic entry of antigens during cross-presentation.One-Sentence SummaryPore-forming protein perforin-2 is a dedicated effector of endocytic escape specific to cross-presenting cells</jats:sec

Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes