Biology subjects

Schulman, B. A.

Publications and source records attributed to Schulman, B. A..

24 records · Page 2Linked to original sources

bioRxiv2020

DIA-based systems biology approach unveils novel E3-dependent responses to a metabolic shift

Yeast Saccharomyces cerevisiae is a powerful model system for systems-wide biology screens and large-scale proteomics methods. Nearly complete proteomics coverage has been achieved owing to advances in mass spectrometry. However, it remains challenging to scale this technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology workflow employing plate-based sample preparation and rapid, single-run data independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement and enables quantitative profiling and comparisons of hundreds of nearly complete yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to environmental perturbations, identifying distinct responses to each of them, and providing a comprehensive resource of these responses. Apart from rapidly recapitulating previously observed responses, we characterized carbon source dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. This unveiled new regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system read-out pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation novel targets of the GID E3 ligase. Moreover, our approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation. Although developed in yeast, rapid whole proteome-based readouts can serve as comprehensive systems-level assay in all cellular systems.

systems biology↗

bioRxiv2020

Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology

Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. We developed a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identified 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells - double the number and quantitative accuracy of data dependent acquisition. Applied to TNF-alpha signaling, the workflow comprehensively captured known sites while adding many novel ones. A first systems-wide investigation of ubiquitination of the circadian cycle uncovered hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting novel connections between metabolism and circadian regulation.

systems biology↗

bioRxiv2020

A selective autophagy pathway for phase separated endocytic protein deposits

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargoes to the membrane bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we discover a selective autophagy pathway for protein condensates formed by endocytic proteins. In this pathway, the endocytic yeast protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis, does not involve other known selective autophagy receptors, but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates - at the plasma membrane and in autophagic bodies - shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy pathway. Our data suggest a model for autophagic degradation of membraneless compartments by the action of intrinsic autophagy receptors. HighlightsO_LIEde1 is a selective autophagy receptor for aberrant CME protein assemblies C_LIO_LIAberrant CME assemblies form by liquid-liquid phase separation C_LIO_LICore autophagy machinery and Ede1 are important for degradation of CME condensates C_LIO_LIUltrastrucural view of a LLPS compartment at the PM and within autophagic bodies C_LI

cell biology↗

bioRxiv2020

Integrative proteomics reveals principles of dynamic phospho-signaling networks in human erythropoiesis

Human erythropoiesis is exquisitely controlled at multiple levels and its dysregulation leads to numerous human diseases. Despite many functional studies focused on classical regulators, we lack a global, system-wide understanding of post-translational mechanisms coordinating erythroid maturation. Using the latest advances in mass spectrometry (MS)-based proteomics we comprehensively investigate the dynamics of protein and post-translational regulation of in vitro reconstituted CD34+ HSPC-derived erythropoiesis. This quantifies and dynamically tracks 7,400 proteins and 27,000 phosphorylation sites. Our data reveals differential temporal protein expression encompassing most protein classes and numerous post-translational regulatory cascades. Drastic cell surface remodeling across erythropoiesis include numerous orchestrated changes in solute carriers, providing new stage-specific markers. The dynamic phosphoproteomes combined with a kinome-targeting CRISPR/Cas9 screen reveal coordinated networks of erythropoietic kinases and downregulation of MAPK signaling subsequent to c-Kit attenuation as key drivers of maturation. Our global view of erythropoiesis establishes a central role of post-translational regulation in terminal differentiation.

systems biology↗

bioRxiv2019

Interconversion between Anticipatory and Active GID E3 Ubiquitin Ligase Conformations via Metabolically Driven Substrate Receptor Assembly

Cells respond to environmental changes by toggling metabolic pathways, preparing for homeostasis, and anticipating future stresses. For example, in Saccharomyces cerevisiae, carbon stress-induced gluconeogenesis is terminated upon glucose availability, a process that involves the multiprotein E3 ligase, GIDSR4, recruiting N-termini and catalyzing ubiquitylation of gluconeogenic enzymes. Here, genetics, biochemistry, and cryo electron microscopy define molecular underpinnings of glucose-induced degradation. Unexpectedly, carbon stress induces an inactive anticipatory complex (GIDAnt), which awaits a glucose-induced substrate receptor to form the active GIDSR4. Meanwhile, other environmental perturbations elicit production of an alternative substrate receptor assembling into a related E3 ligase complex. The intricate structure of GIDAnt enables anticipating and ultimately binding various N-degron targeting (i.e. \"N-end rule\") substrate receptors, while the GIDSR4 E3 forms a clamp-like structure juxtaposing substrate lysines with the ubiquitylation active site. The data reveal evolutionarily conserved GID complexes as a family of multisubunit E3 ubiquitin ligases responsive to extracellular stimuli.

molecular biology↗

bioRxiv2019

A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

Ubiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitins C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at \"exosites\". However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.\n\nSIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

biochemistry↗