SnapShot: Clinical proteomics.

In-depth quantitative proteomic analysis of clinical specimens offers unique and invaluable insights into their protein composition and biochemical state in physiological and pathological conditions. A plethora of methodologies have been developed and customized. Here, we summarize the most common and emerging mass spectrometry (MS)-based workflows. Due to space limitations the overview cannot be complete. To view this SnapShot, open or download the PDF.

Disentangling Genetic and Environmental Effects on the Proteotypes of Individuals.

Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.

The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis.

Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction in vivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

A Class of Environmental and Endogenous Toxins Induces BRCA2 Haploinsufficiency and Genome Instability.

Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.

On the Dependency of Cellular Protein Levels on mRNA Abundance.

The question of how genomic information is expressed to determine phenotypes is of central importance for basic and translational life science research and has been studied by transcriptomic and proteomic profiling. Here, we review the relationship between protein and mRNA levels under various scenarios, such as steady state, long-term state changes, and short-term adaptation, demonstrating the complexity of gene expression regulation, especially during dynamic transitions. The spatial and temporal variations of mRNAs, as well as the local availability of resources for protein biosynthesis, strongly influence the relationship between protein levels and their coding transcripts. We further discuss the buffering of mRNA fluctuations at the level of protein concentrations. We conclude that transcript levels by themselves are not sufficient to predict protein levels in many scenarios and to thus explain genotype-phenotype relationships and that high-quality data quantifying different levels of gene expression are indispensable for the complete understanding of biological processes.

Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation.

Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.

Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome.

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

Molecular architecture of the 40S⋠eIF1⋠eIF3 translation initiation complex.

Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population.

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation.

Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling.

Cytosolic compartmentalization through liquid-liquid unmixing, such as the formation of RNA granules, is involved in many cellular processes and might be used to regulate signal transduction. However, specific molecular mechanisms by which liquid-liquid unmixing and signal transduction are coupled remain unknown. Here, we show that during cellular stress the dual specificity kinase DYRK3 regulates the stability of P-granule-like structures and mTORC1 signaling. DYRK3 displays a cyclic partitioning mechanism between stress granules and the cytosol via a low-complexity domain in its N terminus and its kinase activity. When DYRK3 is inactive, it prevents stress granule dissolution and the release of sequestered mTORC1. When DYRK3 is active, it allows stress granule dissolution, releasing mTORC1 for signaling and promoting its activity by directly phosphorylating the mTORC1 inhibitor PRAS40. This mechanism links cytoplasmic compartmentalization via liquid phase transitions with cellular signaling.

Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.

The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.

Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex.

INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.

Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases.

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.

Mass spectrometry-based proteomics and network biology.

In the life sciences, a new paradigm is emerging that places networks of interacting molecules between genotype and phenotype. These networks are dynamically modulated by a multitude of factors, and the properties emerging from the network as a whole determine observable phenotypes. This paradigm is usually referred to as systems biology, network biology, or integrative biology. Mass spectrometry (MS)-based proteomics is a central life science technology that has realized great progress toward the identification, quantification, and characterization of the proteins that constitute a proteome. Here, we review how MS-based proteomics has been applied to network biology to identify the nodes and edges of biological networks, to detect and quantify perturbation-induced network changes, and to correlate dynamic network rewiring with the cellular phenotype. We discuss future directions for MS-based proteomics within the network biology paradigm.

DIP-MS: ultra-deep interaction proteomics for the deconvolution of protein complexes.

Most proteins are organized in macromolecular assemblies, which represent key functional units regulating and catalyzing most cellular processes. Affinity purification of the protein of interest combined with liquid chromatography coupled to tandem mass spectrometry (AP-MS) represents the method of choice to identify interacting proteins. The composition of complex isoforms concurrently present in the AP sample can, however, not be resolved from a single AP-MS experiment but requires computational inference from multiple time- and resource-intensive reciprocal AP-MS experiments. Here we introduce deep interactome profiling by mass spectrometry (DIP-MS), which combines AP with blue-native-PAGE separation, data-independent acquisition with mass spectrometry and deep-learning-based signal processing to resolve complex isoforms sharing the same bait protein in a single experiment. We applied DIP-MS to probe the organization of the human prefoldin family of complexes, resolving distinct prefoldin holo- and subcomplex variants, complex-complex interactions and complex isoforms with new subunits that were experimentally validated. Our results demonstrate that DIP-MS can reveal proteome modularity at unprecedented depth and resolution.

Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells.

Data on absolute molecule numbers will empower the modeling, understanding, and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under two key physiological conditions. The integrated data set supports quantitative biology and affords unique insights into cell regulation. Although mRNAs are typically expressed in a narrow range above 1 copy/cell, most long, noncoding RNAs, except for a distinct subset, are tightly repressed below 1 copy/cell. Cell-cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also bring about more switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.