A comprehensive landscape of the zinc-regulated human proteome.

Zinc is an essential micronutrient that regulates a wide range of physiological processes, principally through Zn 2+ binding to protein cysteine residues. Despite being critical for modulation of protein function, for the vast majority of the human proteome the cysteine sites subject to regulation by Zn 2+ binding remain undefined. Here we develop ZnCPT, a comprehensive and quantitative mapping of the zinc-regulated cysteine proteome. We define 4807 zinc-regulated protein cysteines, uncovering protein families across major domains of biology that are subject to either constitutive or inducible modification by zinc. ZnCPT enables systematic discovery of zinc-regulated structural, enzymatic, and allosteric functional domains. On this basis, we identify 52 cancer genetic dependencies subject to zinc regulation, and nominate malignancies sensitive to zinc-induced cytotoxicity. In doing so, we discover a mechanism of zinc regulation over Glutathione Reductase (GSR) that drives cell death in GSR-dependent lung cancers. We provide ZnCPT as a resource for understanding mechanisms of zinc regulation over protein function.

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor.

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

Gasdermin D pore-forming activity is redox-sensitive.

Reactive oxygen species (ROS) regulate the activities of inflammasomes, which are innate immune signaling organelles that induce pyroptosis. The mechanisms by which ROS control inflammasome activities are unclear and may be multifaceted. Herein, we report that the protein gasdermin D (GSDMD), which forms membrane pores upon cleavage by inflammasome-associated caspases, is a direct target of ROS. Exogenous and endogenous sources of ROS, and ROS-inducing stimuli that prime cells for pyroptosis induction, promote oligomerization of cleaved GSDMD, leading to membrane rupture and cell death. We find that ROS enhance GSDMD activities through oxidative modification of cysteine 192 (C192). Within macrophages, GSDMD mutants lacking C192 show impaired ability to form membrane pores and induce pyroptosis. Reciprocal mutagenesis studies reveal that C192 is the only cysteine within GSDMD that mediates ROS responsiveness. Cellular redox state is therefore a key determinant of GSDMD activities.

Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and O-GalNAc (the Tn Antigen) in Human Cancer Cells.

Glycoproteins with diverse glycans are essential to human cells, and subtle differences in glycan structures may result in entirely different functions. One typical example is proteins modified with O-linked ß-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc) (the Tn antigen), in which the two glycans have very similar structures and identical chemical compositions, making them extraordinarily challenging to be distinguished. Here, we developed an effective method benefiting from selective enrichment and the enzymatic specificity to simultaneously identify and distinguish glycoproteins with O-GlcNAc and O-GalNAc. Metabolic labeling was combined with bioorthogonal chemistry for enriching glycoproteins modified with O-GlcNAc and O-GalNAc. Then, the enzymatic reaction with galactose oxidase was utilized to specifically oxidize O-GalNAc, but not O-GlcNAc, generating the different tags between glycopeptides with O-GlcNAc and O-GalNAc that can be easily distinguishable by mass spectrometry (MS). Among O-GlcNAcylated proteins commonly identified in three types of human cells, those related to transcription and RNA binding are highly enriched. Cell-specific features are also revealed. Among glycoproteins exclusively in Jurkat cells, those involved in human T-lymphotropic virus type 1 (HTLV-1) infection are overrepresented, which is consistent with the cell line source and suggests that protein O-GlcNAcylation participated in the response to the virus infection. Furthermore, glycoproteins with the Tn antigen have different subcellular distributions in different cells, which may be attributed to the distinct mechanisms for the formation of protein O-GalNAcylation.

Cysteine 253 of UCP1 regulates energy expenditure and sex-dependent adipose tissue inflammation.

Uncoupling protein 1 (UCP1) is a major regulator of brown and beige adipocyte energy expenditure and metabolic homeostasis. However, the widely employed UCP1 loss-of-function model has recently been shown to have a severe deficiency in the entire electron transport chain of thermogenic fat. As such, the role of UCP1 in metabolic regulation in vivo remains unclear. We recently identified cysteine-253 as a regulatory site on UCP1 that elevates protein activity upon covalent modification. Here, we examine the physiological importance of this site through the generation of a UCP1 cysteine-253-null (UCP1 C253A) mouse, a precise genetic model for selective disruption of UCP1 in vivo. UCP1 C253A mice exhibit significantly compromised thermogenic responses in both males and females but display no measurable effect on fat accumulation in an obesogenic environment. Unexpectedly, we find that a lack of C253 results in adipose tissue redox stress, which drives substantial immune cell infiltration and systemic inflammatory pathology in adipose tissues and liver of male, but not female, mice. Elevation of systemic estrogen reverses this male-specific pathology, providing a basis for protection from inflammation due to loss of UCP1 C253 in females. Together, our results establish the UCP1 C253 activation site as a regulator of acute thermogenesis and sex-dependent tissue inflammation.

Sample multiplexing for targeted pathway proteomics in aging mice.

Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over targeted sample multiplexing experiments, termed Tomahto, and present its implementation on the Orbitrap Tribrid mass spectrometer platform. Importantly, this software monitors via the external desktop computer to the data stream and inserts optimized MS2 and MS3 scans in real time based on an application programming interface with the mass spectrometer. Hundreds of proteins of interest from diverse biological samples can be targeted and accurately quantified in a sensitive and high-throughput fashion. It achieves sensitivity comparable to, if not better than, deep fractionation and requires minimal total sample input (∼10 µg). As a proof-of-principle experiment, we selected four pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (nine tissues from five old and five young mice) to explore effects of aging. Tissue-specific aging is presented here and we highlight the role of inflammation- and metabolism-related processes in white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community.

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.

Systematic quantification of the dynamics of newly synthesized proteins unveiling their degradation pathways in human cells.

Proteins are continuously synthesized during cell growth and proliferation. At the same time, excessive and misfolded proteins have to be degraded, otherwise they are a burden to cells. Protein degradation is essential to maintain proteostasis in cells, and dysfunction of protein degradation systems results in numerous diseases such as cancer and neurodegenerative diseases. Despite the importance of protein degradation, the degradation pathways of many proteins remain to be explored. Here, we comprehensively investigated the degradation of newly synthesized proteins in human cells by integrating metabolic labeling, click chemistry, and multiplexed proteomics, and systematic and quantitative analysis of newly synthesized proteins first revealed the degradation pathways of many proteins. Bioinformatic analysis demonstrates that proteins degraded through two major pathways have distinct properties and functions. Proteins degraded through the ubiquitin-proteasome pathway contain more disordered structures, whereas those through the autophagy-lysosome pathway have significantly higher hydrophobicity. Systematic and quantitative investigation of the dynamics of newly synthesized proteins provides unprecedented and valuable information about protein degradation, which leads to a better understanding of protein properties and cellular activities.

Comprehensive Analysis of Protein Glycation Reveals Its Potential Impacts on Protein Degradation and Gene Expression in Human Cells.

Glycation as a type of non-enzymatic protein modification is related to aging and chronic diseases, especially diabetes. Global analysis of protein glycation will aid in a better understanding of its formation mechanism and biological significance. In this work, we comprehensively investigated protein glycation in human cells (HEK293T, Jurkat, and MCF7 cells). The current results indicated that this non-enzymatic modification was not random, and protein at the extracellular regions and the nucleus were more frequently glycated. Systematic and site-specific analysis of glycated proteins allowed us to study the effect of the primary sequences and secondary structures of proteins on glycation. Furthermore, nearly every enzyme in the glycolytic pathway was found to be glycated and a possible mechanism was proposed. Many glycation sites were also previously reported as acetylation and ubiquitination sites, which strongly suggested that this non-enzymatic modification may disturb protein degradation and gene expression. The current results will facilitate further studies of protein glycation in biomedical and clinical research.

Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion.

Human antibody-secreting cells (ASC) in peripheral blood are found after vaccination or infection but rapidly apoptose unless they migrate to the bone marrow (BM). Yet, elements of the BM microenvironment required to sustain long-lived plasma cells (LLPC) remain elusive. Here, we identify BM factors that maintain human ASC > 50 days in vitro. The critical components of the cell-free in vitro BM mimic consist of products from primary BM mesenchymal stromal cells (MSC), a proliferation-inducing ligand (APRIL), and hypoxic conditions. Comparative analysis of protein-protein interactions between BM-MSC proteomics with differential RNA transcriptomics of blood ASC and BM LLPC identify two major survival factors, fibronectin and YWHAZ. The MSC secretome proteins and hypoxic conditions play a role in LLPC survival utilizing mechanisms that downregulate mTORC1 signaling and upregulate hypoxia signatures. In summary, we identify elements of the BM survival niche critical for maturation of blood ASC to BM LLPC.

Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods.

Protein N-glycosylation is essential for mammalian cell survival and is well-known to be involved in many biological processes. Aberrant glycosylation is directly related to human disease including cancer and infectious diseases. Global analysis of protein N-glycosylation will allow a better understanding of protein functions and cellular activities. Mass spectrometry (MS)-based proteomics provides a unique opportunity to site-specifically characterize protein glycosylation on a large scale. Due to the complexity of biological samples, effective enrichment methods are critical prior to MS analysis. Here, we compared two lectin-independent methods to enrich glycopeptides for the global analysis of protein N-glycosylation by MS. The first boronic acid-based enrichment (BA) method benefits from the universal and reversible interactions between boronic acid and sugars; the other method utilizes metabolic labeling and click chemistry (MC) to incorporate a chemical handle into glycoproteins for future affinity enrichment. We comprehensively compared the performance of the two methods in the identification and quantification of glycoproteins in statin-treated liver cells. Based on the current results, the BA method is more universal in enriching glycopeptides, while with the MC method, cell surface glycoproteins were highly enriched, and the quantification results appear to be more dynamic because only the newly-synthesized glycoproteins were analyzed. In addition, we normalized the glycosylation site ratios by the corresponding parent protein ratios to reflect the real modification changes. In combination with MS-based proteomics, effective enrichment methods will vertically advance protein glycosylation research.

Mass Spectrometry-Based Chemical and Enzymatic Methods for Global Analysis of Protein Glycosylation.

Glycosylation is one of the most common protein modifications, and it is essential for mammalian cell survival. It often determines protein folding and trafficking, and regulates nearly every extracellular activity, including cell-cell communication and cell-matrix interactions. Aberrant protein glycosylation events are hallmarks of human diseases such as cancer and infectious diseases. Therefore, glycoproteins can serve as effective biomarkers for disease detection and targets for drug and vaccine development. Despite the importance of glycoproteins, global analysis of protein glycosylation (either glycoproteins or glycans) in complex biological samples has been a daunting task, and here we mainly focus on glycoprotein analysis using mass spectrometry (MS)-based bottom-up proteomics. Although the emergence of MS-based proteomics has provided a great opportunity to analyze glycoproteins globally, the low abundance of many glycoproteins and the heterogeneity of glycans dramatically increase the technical difficulties. In order to overcome these obstacles, considerable progress has been made in recent years, which has contributed to comprehensive analysis of glycoproteins. In our lab, we developed effective MS-based chemical and enzymatic methods to (1) globally analyze glycoproteins in complex biological samples, (2) target glycoproteins specifically on the surface of human cells, (3) systematically quantify glycoprotein and surface glycoprotein dynamics (the abundance changes of glycoproteins as a function of time), and (4) selectively characterize glycoproteins with a particular and important glycan. In this Account, we first briefly describe the glycopeptide/protein enrichment methods in the literature and then discuss the developments of boronic acid-based methods to enrich glycopeptides for large-scale analysis of protein glycosylation. Boronic acids can form reversible covalent interactions with sugars, but the low binding affinity of normal boronic acid-based methods prevents us from capturing glycoproteins with low abundance, which often contain more valuable information. We enhanced the boronic acid-glycan interactions by using a boronic acid derivative (benzoboroxole) and conjugating it onto a dendrimer to allow synergistic interactions between the boronic acid derivative and sugars. The new method is capable of globally analyzing protein glycosylation with site and glycan structure information, especially for those with low abundance. In the next part, we discuss the combination of metabolic labeling, click chemistry and enzymatic reactions, and MS-based proteomics as a very powerful approach for surface glycoproteome analysis in human cells. The methods enable us to specifically identify surface glycoproteins and to quantify their abundance changes and dynamics together with quantitative proteomics. The last section of this Account focuses on chemical and enzymatic methods to study glycoproteins containing a particular and important glycan (the Tn antigen, i.e., O-GalNAc). Although not comprehensive, this Account provides an overview of chemical and enzymatic methods to characterize protein glycosylation in combination with MS-based proteomics. These methods will have extensive applications in the fields of biology and biomedicine, which will lead to a better understanding of glycoprotein functions and the molecular mechanisms of diseases. Eventually, glycoproteins will be identified as effective biomarkers for disease detection and drug targets for disease treatment.

An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins.

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. However, the heterogeneity of glycans and the low abundance of many glycoproteins complicate their global analysis. Chemical methods based on reversible covalent interactions between boronic acid and glycans have great potential to enrich glycopeptides, but the binding affinity is typically not strong enough to capture low-abundance species. Here, we develop a strategy using dendrimer-conjugated benzoboroxole to enhance the glycopeptide enrichment. We test the performance of several boronic acid derivatives, showing that benzoboroxole markedly increases glycopeptide coverage from human cell lysates. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole-glycan interactions. This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. Importantly, the enriched glycopeptides remain intact, making the current method compatible with mass-spectrometry-based approaches to identify glycosylation sites and glycan structures.

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.

Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, incomplete reactions and side reactions may occur, which compromise the experimental results. In this work, we systematically evaluated the reduction step with commonly used reagents, i.e., dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, or tris(3-hydroxypropyl)phosphine, and alkylation with iodoacetamide, acrylamide, N-ethylmaleimide, or 4-vinylpyridine. By using digested peptides from a yeast whole-cell lysate, the number of proteins and peptides identified were very similar using four different reducing reagents. The results from four alkylating reagents, however, were dramatically different with iodoacetamide giving the highest number of peptides with alkylated cysteine and the lowest number of peptides with incomplete cysteine alkylation and side reactions. Alkylation conditions with iodoacetamide were further optimized. To identify more peptides with cysteine, thiopropyl-sepharose 6B resins were used to enrich them, and the optimal conditions were employed for the reduction and alkylation. The enrichment resulted in over three times more cysteine-containing peptides than without enrichment. Systematic evaluation of the reduction and alkylation with different reagents can aid in a better design of bottom-up proteomic experiments.

Simultaneous Quantitation of Glycoprotein Degradation and Synthesis Rates by Integrating Isotope Labeling, Chemical Enrichment, and Multiplexed Proteomics.

Protein glycosylation is essential for cell survival and regulates many cellular events. Reversible glycosylation is also dynamic in biological systems. The functions of glycoproteins are regulated by their dynamics to adapt the ever-changing inter- and intracellular environments. Glycans on proteins not only mediate a variety of protein activities, but also creates a steric hindrance for protecting the glycoproteins from degradation by proteases. In this work, a novel strategy integrating isotopic labeling, chemical enrichment and multiplexed proteomics was developed to simultaneously quantify the degradation and synthesis rates of many glycoproteins in human cells. We quantified the synthesis rates of 847 N-glycoproteins and the degradation rates of 704 glycoproteins in biological triplicate experiments, including many important glycoproteins such as CD molecules. Through comparing the synthesis and degradation rates, we found that most proteins have higher synthesis rates since cells are still growing throughout the time course, while a small group of proteins with lower synthesis rates mainly participate in adhesion, locomotion, localization, and signaling. This method can be widely applied in biochemical and biomedical research and provide insights into elucidating glycoprotein functions and the molecular mechanism of many biological events.

Evidence for the importance of post-transcriptional regulatory changes in ovarian cancer progression and the contribution of miRNAs.

High-throughput technologies have identified significant changes in patterns of mRNA expression over cancer development but the functional significance of these changes often rests upon the assumption that observed changes in levels of mRNA accurately reflect changes in levels of their encoded proteins. We systematically compared the expression of 4436 genes on the RNA and protein levels between discrete tumor samples collected from the ovary and from the omentum of the same OC patient. The overall correlation between global changes in levels of mRNA and their encoding proteins is low (r = 0.38). The majority of differences are on the protein level with no corresponding change on the mRNA level. Indirect and direct evidence indicates that a significant fraction of the differences may be mediated by microRNAs.

Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins.

Metastasis is responsible for most cancer-related deaths, but the current clinical treatments are not effective. Recently, gold nanoparticles (AuNPs) were discovered to inhibit cancer cell migration and prevent metastasis. Rationally designed AuNPs could greatly benefit their antimigration property, but the molecular mechanisms need to be explored. Cytoskeletons are cell structural proteins that closely relate to migration, and surface receptor integrins play critical roles in controlling the organization of cytoskeletons. Herein, we developed a strategy to inhibit cancer cell migration by targeting integrins, using Arg-Gly-Asp (RGD) peptide-functionalized gold nanorods. To enhance the effect, AuNRs were further activated with 808-nm near-infrared (NIR) light to generate heat for photothermal therapy (PPTT), where the temperature was adjusted not to affect the cell viability/proliferation. Our results demonstrate changes in cell morphology, observed as cytoskeleton protrusions-i.e., lamellipodia and filopodia-were reduced after treatment. The Western blot analysis indicates the downstream effectors of integrin were attracted toward the antimigration direction. Proteomics results indicated broad perturbations in four signaling pathways, Rho GTPases, actin, microtubule, and kinases-related pathways, which are the downstream regulators of integrins. Due to the dominant role of integrins in controlling cytoskeleton, focal adhesion, actomyosin contraction, and actin and microtubule assembly have been disrupted by targeting integrins. PPTT further enhanced the remodeling of cytoskeletal proteins and decreased migration. In summary, the ability of targeting AuNRs to cancer cell integrins and the introduction of PPTT stimulated broad regulation on the cytoskeleton, which provides the evidence for a potential medical application for controlling cancer metastasis.

Specific Identification of Glycoproteins Bearing the Tn Antigen in Human Cells.

Glycoproteins contain a wealth of valuable information regarding the development and disease status of cells. In cancer cells, some glycans (such as the Tn antigen) are highly up-regulated, but this remains largely unknown for glycoproteins with a particular glycan. Herein, an innovative method combining enzymatic and chemical reactions was first designed to enrich glycoproteins with the Tn antigen. Using synthetic glycopeptides with O-GalNAc (the Tn antigen) or O-GlcNAc, we demonstrated that the method is selective for glycopeptides with O-GalNAc and can distinguish between these two modifications. The diagnostic ions from the tagged O-GalNAc further confirmed the effectiveness of the method and confidence in the identification of glycopeptides with the Tn antigen by mass spectrometry. Using this method, we identified 96 glycoproteins with the Tn antigen in Jurkat cells. The method can be extensively applied in biological and biomedical research.

Efficacy, long-term toxicity, and mechanistic studies of gold nanorods photothermal therapy of cancer in xenograft mice.

Gold nanorods (AuNRs)-assisted plasmonic photothermal therapy (AuNRs-PPTT) is a promising strategy for combating cancer in which AuNRs absorb near-infrared light and convert it into heat, causing cell death mainly by apoptosis and/or necrosis. Developing a valid PPTT that induces cancer cell apoptosis and avoids necrosis in vivo and exploring its molecular mechanism of action is of great importance. Furthermore, assessment of the long-term fate of the AuNRs after treatment is critical for clinical use. We first optimized the size, surface modification [rifampicin (RF) conjugation], and concentration (2.5 nM) of AuNRs and the PPTT laser power (2 W/cm2) to achieve maximal induction of apoptosis. Second, we studied the potential mechanism of action of AuNRs-PPTT using quantitative proteomic analysis in mouse tumor tissues. Several death pathways were identified, mainly involving apoptosis and cell death by releasing neutrophil extracellular traps (NETs) (NETosis), which were more obvious upon PPTT using RF-conjugated AuNRs (AuNRs@RF) than with polyethylene glycol thiol-conjugated AuNRs. Cytochrome c and p53-related apoptosis mechanisms were identified as contributing to the enhanced effect of PPTT with AuNRs@RF. Furthermore, Pin1 and IL18-related signaling contributed to the observed perturbation of the NETosis pathway by PPTT with AuNRs@RF. Third, we report a 15-month toxicity study that showed no long-term toxicity of AuNRs in vivo. Together, these data demonstrate that our AuNRs-PPTT platform is effective and safe for cancer therapy in mouse models. These findings provide a strong framework for the translation of PPTT to the clinic.