Metalloproteomics for Biomedical Research: Methodology and Applications.

Metals are essential components in life processes and participate in many important biological processes. Dysregulation of metal homeostasis is correlated with many diseases. Metals are also frequently incorporated into diagnosis and therapeutics. Understanding of metal homeostasis under (patho)physiological conditions and the molecular mechanisms of action of metallodrugs in biological systems has positive impacts on human health. As an emerging interdisciplinary area of research, metalloproteomics involves investigating metal-protein interactions in biological systems at a proteome-wide scale, has received growing attention, and has been implemented into metal-related research. In this review, we summarize the recent advances in metalloproteomics methodologies and applications. We also highlight emerging single-cell metalloproteomics, including time-resolved inductively coupled plasma mass spectrometry, mass cytometry, and secondary ion mass spectrometry. Finally, we discuss future perspectives in metalloproteomics, aiming to attract more original research to develop more advanced methodologies, which could be utilized rapidly by biochemists or biologists to expand our knowledge of how metal functions in biology and medicine.

Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.

Single-Molecule Studies of Protein Folding with Optical Tweezers.

Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.

How Does the Ribosome Fold the Proteome?

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.

Chemical Biology Framework to Illuminate Proteostasis.

Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.

A Quantitative Proteome Map of the Human Body.

Determining protein levels in each tissue and how they compare with RNA levels is important for understanding human biology and disease as well as regulatory processes that control protein levels. We quantified the relative protein levels from over 12,000 genes across 32 normal human tissues. Tissue-specific or tissue-enriched proteins were identified and compared to transcriptome data. Many ubiquitous transcripts are found to encode tissue-specific proteins. Discordance of RNA and protein enrichment revealed potential sites of synthesis and action of secreted proteins. The tissue-specific distribution of proteins also provides an in-depth view of complex biological events that require the interplay of multiple tissues. Most importantly, our study demonstrated that protein tissue-enrichment information can explain phenotypes of genetic diseases, which cannot be obtained by transcript information alone. Overall, our results demonstrate how understanding protein levels can provide insights into regulation, secretome, metabolism, and human diseases.

A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging.

Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

Proteogenomic Characterization of Endometrial Carcinoma.

We undertook a comprehensive proteogenomic characterization of 95 prospectively collected endometrial carcinomas, comprising 83 endometrioid and 12 serous tumors. This analysis revealed possible new consequences of perturbations to the p53 and Wnt/ß-catenin pathways, identified a potential role for circRNAs in the epithelial-mesenchymal transition, and provided new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. An extensive genome-wide acetylation survey yielded insights into regulatory mechanisms linking Wnt signaling and histone acetylation. We also characterized aspects of the tumor immune landscape, including immunogenic alterations, neoantigens, common cancer/testis antigens, and the immune microenvironment, all of which can inform immunotherapy decisions. Collectively, our multi-omic analyses provide a valuable resource for researchers and clinicians, identify new molecular associations of potential mechanistic significance in the development of endometrial cancers, and suggest novel approaches for identifying potential therapeutic targets.

Effects of aneuploidy on cell behaviour and function.

Aneuploidy, a genomic alternation characterized by deviations in the copy number of chromosomes, affects organisms from early development through to aging. Although it is a main cause of human pregnancy loss and a hallmark of cancer, how aneuploidy affects cellular function has been elusive. The last two decades have seen rapid advances in the understanding of the causes and consequences of aneuploidy at the molecular and cellular levels. These studies have uncovered effects of aneuploidy that can be beneficial or detrimental to cells and organisms in an environmental context-dependent and karyotype-dependent manner. Aneuploidy also imposes general stress on cells that stems from an imbalanced genome and, consequently, also an imbalanced proteome. These insights provide the fundamental framework for understanding the impact of aneuploidy in genome evolution, human pathogenesis and drug resistance.

Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation.

Bioactive molecules can pass between microbiota and host to influence host cellular functions. However, general principles of interspecies communication have not been discovered. We show here in C. elegans that nitric oxide derived from resident bacteria promotes widespread S-nitrosylation of the host proteome. We further show that microbiota-dependent S-nitrosylation of C. elegans Argonaute protein (ALG-1)-at a site conserved and S-nitrosylated in mammalian Argonaute 2 (AGO2)-alters its function in controlling gene expression via microRNAs. By selectively eliminating nitric oxide generation by the microbiota or S-nitrosylation in ALG-1, we reveal unforeseen effects on host development. Thus, the microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development. Our findings suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.

CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

Proteomic and interactomic insights into the molecular basis of cell functional diversity.

The ability of living systems to adapt to changing conditions originates from their capacity to change their molecular constitution. This is achieved by multiple mechanisms that modulate the quantitative composition and the diversity of the molecular inventory. Molecular diversification is particularly pronounced on the proteome level, at which multiple proteoforms derived from the same gene can in turn combinatorially form different protein complexes, thus expanding the repertoire of functional modules in the cell. The study of molecular and modular diversity and their involvement in responses to changing conditions has only recently become possible through the development of new 'omics'-based screening technologies. This Review explores our current knowledge of the mechanisms regulating functional diversification along the axis of gene expression, with a focus on the proteome and interactome. We explore the interdependence between different molecular levels and how this contributes to functional diversity. Finally, we highlight several recent techniques for studying molecular diversity, with specific focus on mass spectrometry-based analysis of the proteome and its organization into functional modules, and examine future directions for this rapidly growing field.

The proteomic landscape of genotoxic stress-induced micronuclei.

Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.

Spatial proteomics: a powerful discovery tool for cell biology.

Protein subcellular localization is tightly controlled and intimately linked to protein function in health and disease. Capturing the spatial proteome - that is, the localizations of proteins and their dynamics at the subcellular level - is therefore essential for a complete understanding of cell biology. Owing to substantial advances in microscopy, mass spectrometry and machine learning applications for data analysis, the field is now mature for proteome-wide investigations of spatial cellular regulation. Studies of the human proteome have begun to reveal a complex architecture, including single-cell variations, dynamic protein translocations, changing interaction networks and proteins localizing to multiple compartments. Furthermore, several studies have successfully harnessed the power of comparative spatial proteomics as a discovery tool to unravel disease mechanisms. We are at the beginning of an era in which spatial proteomics finally integrates with cell biology and medical research, thereby paving the way for unbiased systems-level insights into cellular processes. Here, we discuss current methods for spatial proteomics using imaging or mass spectrometry and specifically highlight global comparative applications. The aim of this Review is to survey the state of the field and also to encourage more cell biologists to apply spatial proteomics approaches.

Establishing and regulating the composition of cilia for signal transduction.

The primary cilium is a hair-like surface-exposed organelle of the eukaryotic cell that decodes a variety of signals - such as odorants, light and Hedgehog morphogens - by altering the local concentrations and activities of signalling proteins. Signalling within the cilium is conveyed through a diverse array of second messengers, including conventional signalling molecules (such as cAMP) and some unusual intermediates (such as sterols). Diffusion barriers at the ciliary base establish the unique composition of this signalling compartment, and cilia adapt their proteome to signalling demands through regulated protein trafficking. Much progress has been made on the molecular understanding of regulated ciliary trafficking, which encompasses not only exchanges between the cilium and the rest of the cell but also the shedding of signalling factors into extracellular vesicles.

Structured States of Disordered Proteins from Genomic Sequences.

Protein flexibility ranges from simple hinge movements to functional disorder. Around half of all human proteins contain apparently disordered regions with little 3D or functional information, and many of these proteins are associated with disease. Building on the evolutionary couplings approach previously successful in predicting 3D states of ordered proteins and RNA, we developed a method to predict the potential for ordered states for all apparently disordered proteins with sufficiently rich evolutionary information. The approach is highly accurate (79%) for residue interactions as tested in more than 60 known disordered regions captured in a bound or specific condition. Assessing the potential for structure of more than 1,000 apparently disordered regions of human proteins reveals a continuum of structural order with at least 50% with clear propensity for three- or two-dimensional states. Co-evolutionary constraints reveal hitherto unseen structures of functional importance in apparently disordered proteins.

Illuminating the Dark Proteome.

A large segment of the proteome consists of disordered regions, yet in most cases, little is known about their mechanisms and functions. What are the roles of protein disorder in cell biology, and how do intrinsically disordered proteins function? These are the questions Cell's Robert Kruger posed to Madan Babu, Julie Forman-Kay, and Richard Kriwacki. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online. PAPERCLIP.

Base editor screens for in situ mutational scanning at scale.

A fundamental challenge in biology is understanding the molecular details of protein function. How mutations alter protein activity, regulation, and response to drugs is of critical importance to human health. Recent years have seen the emergence of pooled base editor screens for in situ mutational scanning: the interrogation of protein sequence-function relationships by directly perturbing endogenous proteins in live cells. These studies have revealed the effects of disease-associated mutations, discovered novel drug resistance mechanisms, and generated biochemical insights into protein function. Here, we discuss how this "base editor scanning" approach has been applied to diverse biological questions, compare it with alternative techniques, and describe the emerging challenges that must be addressed to maximize its utility. Given its broad applicability toward profiling mutations across the proteome, base editor scanning promises to revolutionize the investigation of proteins in their native contexts.