Haopeng Xiao

All Publications

• Mitochondrial complex III-derived ROS amplify immunometabolic changes in astrocytes and promote dementia pathology. bioRxiv : the preprint server for biology Barnett, D., Zimmer, T. S., Booraem, C., Palaguachi, F., Meadows, S. M., Xiao, H., Chouchani, E. T., Orr, A. G., Orr, A. L. 2024

  Abstract

  Neurodegenerative disorders alter mitochondrial functions, including the production of reactive oxygen species (ROS). Mitochondrial complex III (CIII) generates ROS implicated in redox signaling, but its triggers, targets, and disease relevance are not clear. Using site-selective suppressors and genetic manipulations together with mitochondrial ROS imaging and multiomic profiling, we found that CIII is the dominant source of ROS production in astrocytes exposed to neuropathology-related stimuli. Astrocytic CIII-ROS production was dependent on nuclear factor-kappaB (NF-kappaB) and the mitochondrial sodium-calcium exchanger (NCLX) and caused oxidation of select cysteines within immune and metabolism-associated proteins linked to neurological disease. CIII-ROS amplified metabolomic and pathology-associated transcriptional changes in astrocytes, with STAT3 activity as a major mediator, and facilitated neuronal toxicity in a non-cell-autonomous manner. As proof-of-concept, suppression of CIII-ROS in mice decreased dementia-linked tauopathy and neuroimmune cascades and extended lifespan. Our findings establish CIII-ROS as an important immunometabolic signal transducer and tractable therapeutic target in neurodegenerative disease.

  View details for DOI 10.1101/2024.08.19.608708

  View details for PubMedID 39229090

• Parallel control of cold-triggered adipocyte thermogenesis by UCP1 and CKB CELL METABOLISM Rahbani, J. F., Bunk, J., Lagarde, D., Samborska, B., Roesler, A., Xiao, H., Shaw, A., Kaiser, Z., Braun, J. L., Geromella, M. S., Fajardo, V. A., Koza, R. A., Kazak, L. 2024; 36 (3): 526-540.e7

  Abstract

  That uncoupling protein 1 (UCP1) is the sole mediator of adipocyte thermogenesis is a conventional viewpoint that has primarily been inferred from the attenuation of the thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue. To mitigate these potentially confounding ancillary changes, we constructed mice with inducible adipocyte-selective Ucp1 disruption. We find that, although germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with the inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase b (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold intolerance. Following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature brown adipocytes that coordinately restore UCP1 and CKB expression. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy-through UCP1 and CKB-to promote cold-induced energy dissipation.

  View details for DOI 10.1016/j.cmet.2024.01.001

  View details for Web of Science ID 001204590300001

  View details for PubMedID 38272036

• Monocarboxylate transporters facilitate succinate uptake into brown adipocytes. Nature metabolism Reddy, A., Winther, S., Tran, N., Xiao, H., Jakob, J., Garrity, R., Smith, A., Ordonez, M., Laznik-Bogoslavski, D., Rothstein, J. D., Mills, E. L., Chouchani, E. T. 2024; 6 (3): 567-577

  Abstract

  Uptake of circulating succinate by brown adipose tissue (BAT) and beige fat elevates whole-body energy expenditure, counteracts obesity and antagonizes systemic tissue inflammation in mice. The plasma membrane transporters that facilitate succinate uptake in these adipocytes remain undefined. Here we elucidate a mechanism underlying succinate import into BAT via monocarboxylate transporters (MCTs). We show that succinate transport is strongly dependent on the proportion that is present in the monocarboxylate form. MCTs facilitate monocarboxylate succinate uptake, which is promoted by alkalinization of the cytosol driven by adrenoreceptor stimulation. In brown adipocytes, we show that MCT1 primarily facilitates succinate import. In male mice, we show that both acute pharmacological inhibition of MCT1 and congenital depletion of MCT1 decrease succinate uptake into BAT and consequent catabolism. In sum, we define a mechanism of succinate uptake in BAT that underlies its protective activity in mouse models of metabolic disease.

  View details for DOI 10.1038/s42255-024-00981-5

  View details for PubMedID 38378996

  View details for PubMedCentralID 9150600

• Lactate regulates cell cycle by remodeling the anaphase promoting complex. Nature Liu, W., Wang, Y., Bozi, L. H., Fischer, P., Jedrychowski, M. P., Xiao, H., Wu, T., Darabedian, N., He, X., Mills, E. L., Burger, N., Shin, S., Reddy, A., Sprenger, H. G., Tran, N., Winther, S., Hinshaw, S. M., Shen, J., Seo, H. S., Song, K., Xu, A. Z., Sebastian, L., Zhao, J., Dhe-Paganon, S., Che, J., Gygi, S. P., Arthanari, H., Chouchani, E. T. 2023

  Abstract

  Lactate is abundant in rapidly dividing cells due to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here, we deploy a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we elucidate a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodeling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We discover that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. The above mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient replete growth phase to stimulate timed opening of APC/C, cell division, and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodeling and can overcome anti-mitotic pharmacology via mitotic slippage. Taken together, we define a biochemical mechanism through which lactate directly regulates protein function to control cell cycle and proliferation.

  View details for DOI 10.1038/s41586-023-05939-3

  View details for PubMedID 36921622

• Isolation of extracellular fluids reveals novel secreted bioactive proteins from muscle and fat tissues. Cell metabolism Mittenbühler, M. J., Jedrychowski, M. P., Van Vranken, J. G., Sprenger, H. G., Wilensky, S., Dumesic, P. A., Sun, Y., Tartaglia, A., Bogoslavski, D., A, M., Xiao, H., Blackmore, K. A., Reddy, A., Gygi, S. P., Chouchani, E. T., Spiegelman, B. M. 2023; 35 (3): 535-549.e7

  Abstract

  Proteins are secreted from cells to send information to neighboring cells or distant tissues. Because of the highly integrated nature of energy balance systems, there has been particular interest in myokines and adipokines. These are challenging to study through proteomics because serum or plasma contains highly abundant proteins that limit the detection of proteins with lower abundance. We show here that extracellular fluid (EF) from muscle and fat tissues of mice shows a different protein composition than either serum or tissues. Mass spectrometry analyses of EFs from mice with physiological perturbations, like exercise or cold exposure, allowed the quantification of many potentially novel myokines and adipokines. Using this approach, we identify prosaposin as a secreted product of muscle and fat. Prosaposin expression stimulates thermogenic gene expression and induces mitochondrial respiration in primary fat cells. These studies together illustrate the utility of EF isolation as a discovery tool for adipokines and myokines.

  View details for DOI 10.1016/j.cmet.2022.12.014

  View details for PubMedID 36681077

  View details for PubMedCentralID PMC9998376

• Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor. Nature chemical biology Darabedian, N., Ji, W., Fan, M., Lin, S., Seo, H. S., Vinogradova, E. V., Yaron, T. M., Mills, E. L., Xiao, H., Senkane, K., Huntsman, E. M., Johnson, J. L., Che, J., Cantley, L. C., Cravatt, B. F., Dhe-Paganon, S., Stegmaier, K., Zhang, T., Gray, N. S., Chouchani, E. T. 2023

  Abstract

  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.

  View details for DOI 10.1038/s41589-023-01273-x

  View details for PubMedID 36823351

  View details for PubMedCentralID 5540325

• Gasdermin D pore-forming activity is redox-sensitive CELL REPORTS Devant, P., Borsic, E., Ngwa, E. M., Xiao, H., Chouchani, E. T., Thiagarajah, J. R., Hafner-Bratkovic, I., Evavold, C. L., Kagan, J. C. 2023; 42 (1): 112008

  Abstract

  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.

  View details for DOI 10.1016/j.celrep.2023.112008

  View details for Web of Science ID 000924666400001

  View details for PubMedID 36662620

  View details for PubMedCentralID PMC9947919

• Architecture of the outbred brown fat proteome defines regulators of metabolic physiology. Cell Xiao, H., Bozi, L. H., Sun, Y., Riley, C. L., Philip, V. M., Chen, M., Li, J., Zhang, T., Mills, E. L., Emont, M. P., Sun, W., Reddy, A., Garrity, R., Long, J., Becher, T., Vitas, L. P., Laznik-Bogoslavski, D., Ordonez, M., Liu, X., Chen, X., Wang, Y., Liu, W., Tran, N., Liu, Y., Zhang, Y., Cypess, A. M., White, A. P., He, Y., Deng, R., Schöder, H., Paulo, J. A., Jedrychowski, M. P., Banks, A. S., Tseng, Y. H., Cohen, P., Tsai, L. T., Rosen, E. D., Klein, S., Chondronikola, M., McAllister, F. E., Van Bruggen, N., Huttlin, E. L., Spiegelman, B. M., Churchill, G. A., Gygi, S. P., Chouchani, E. T. 2022

  Abstract

  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.

  View details for DOI 10.1016/j.cell.2022.10.003

  View details for PubMedID 36334589

• Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and O-GalNAc (the Tn Antigen) in Human Cancer Cells. Analytical chemistry Xu, S., Zheng, J., Xiao, H., Wu, R. 2022; 94 (7): 3343-3351

  Abstract

  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 β-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.

  View details for DOI 10.1021/acs.analchem.1c05438

  View details for PubMedID 35132862

• Cysteine 253 of UCP1 regulates energy expenditure and sex-dependent adipose tissue inflammation CELL METABOLISM Mills, E. L., Harmon, C., Jedrychowski, M. P., Xiao, H., Gruszczyk, A., Bradshaw, G. A., Tran, N., Garrity, R., Laznik-Bogoslavski, D., Szpyt, J., Prendeville, H., Lynch, L., Murphy, M. P., Gygi, S. P., Spiegelman, B. M., Chouchani, E. T. 2022; 34 (1): 140-+

  Abstract

  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.

  View details for DOI 10.1016/j.cmet.2021.11.003

  View details for Web of Science ID 000740783700014

  View details for PubMedID 34861155

  View details for PubMedCentralID PMC8732317

• Glycogen metabolism links glucose homeostasis to thermogenesis in adipocytes NATURE Keinan, O., Valentine, J. M., Xiao, H., Mahata, S. K., Reilly, S. M., Abu-Odeh, M., Deluca, J. H., Dadpey, B., Cho, L., Pan, A., Yu, R. T., Dai, Y., Liddle, C., Downes, M., Evans, R. M., Lusis, A. J., Laakso, M., Chouchani, E. T., Ryde'n, M., Saltiel, A. R. 2021; 599 (7884): 296-+

  Abstract

  Adipocytes increase energy expenditure in response to prolonged sympathetic activation via persistent expression of uncoupling protein 1 (UCP1)1,2. Here we report that the regulation of glycogen metabolism by catecholamines is critical for UCP1 expression. Chronic β-adrenergic activation leads to increased glycogen accumulation in adipocytes expressing UCP1. Adipocyte-specific deletion of a scaffolding protein, protein targeting to glycogen (PTG), reduces glycogen levels in beige adipocytes, attenuating UCP1 expression and responsiveness to cold or β-adrenergic receptor-stimulated weight loss in obese mice. Unexpectedly, we observed that glycogen synthesis and degradation are increased in response to catecholamines, and that glycogen turnover is required to produce reactive oxygen species leading to the activation of p38 MAPK, which drives UCP1 expression. Thus, glycogen has a key regulatory role in adipocytes, linking glucose metabolism to thermogenesis.

  View details for DOI 10.1038/s41586-021-04019-8

  View details for Web of Science ID 000713338100003

  View details for PubMedID 34707293

  View details for PubMedCentralID PMC9186421

• UCP1 governs liver extracellular succinate and inflammatory pathogenesis. Nature metabolism Mills, E. L., Harmon, C., Jedrychowski, M. P., Xiao, H., Garrity, R., Tran, N. V., Bradshaw, G. A., Fu, A., Szpyt, J., Reddy, A., Prendeville, H., Danial, N. N., Gygi, S. P., Lynch, L., Chouchani, E. T. 2021; 3 (5): 604-617

  Abstract

  Non-alcoholic fatty liver disease (NAFLD), the most prevalent liver pathology worldwide, is intimately linked with obesity and type 2 diabetes. Liver inflammation is a hallmark of NAFLD and is thought to contribute to tissue fibrosis and disease pathogenesis. Uncoupling protein 1 (UCP1) is exclusively expressed in brown and beige adipocytes, and has been extensively studied for its capacity to elevate thermogenesis and reverse obesity. Here we identify an endocrine pathway regulated by UCP1 that antagonizes liver inflammation and pathology, independent of effects on obesity. We show that, without UCP1, brown and beige fat exhibit a diminished capacity to clear succinate from the circulation. Moreover, UCP1KO mice exhibit elevated extracellular succinate in liver tissue that drives inflammation through ligation of its cognate receptor succinate receptor 1 (SUCNR1) in liver-resident stellate cell and macrophage populations. Conversely, increasing brown and beige adipocyte content in mice antagonizes SUCNR1-dependent inflammatory signalling in the liver. We show that this UCP1-succinate-SUCNR1 axis is necessary to regulate liver immune cell infiltration and pathology, and systemic glucose intolerance in an obesogenic environment. As such, the therapeutic use of brown and beige adipocytes and UCP1 extends beyond thermogenesis and may be leveraged to antagonize NAFLD and SUCNR1-dependent liver inflammation.

  View details for DOI 10.1038/s42255-021-00389-5

  View details for PubMedID 34002097

  View details for PubMedCentralID PMC8207988

• IRF3 reduces adipose thermogenesis via ISG15-mediated reprogramming of glycolysis JOURNAL OF CLINICAL INVESTIGATION Yan, S., Kumari, M., Xiao, H., Jacobs, C., Kochumon, S., Jedrychowski, M., Chouchani, E., Ahmad, R., Rosen, E. D. 2021; 131 (7)

  Abstract

  Adipose thermogenesis is repressed in obesity, reducing the homeostatic capacity to compensate for chronic overnutrition. Inflammation inhibits adipose thermogenesis, but little is known about how this occurs. Here we showed that the innate immune transcription factor IRF3 is a strong repressor of thermogenic gene expression and oxygen consumption in adipocytes. IRF3 achieved this by driving expression of the ubiquitin-like modifier ISG15, which became covalently attached to glycolytic enzymes, thus reducing their function and decreasing lactate production. Lactate repletion was able to restore thermogenic gene expression, even when the IRF3/ISG15 axis was activated. Mice lacking ISG15 phenocopied mice lacking IRF3 in adipocytes, as both had elevated energy expenditure and were resistant to diet-induced obesity. These studies provide a deep mechanistic understanding of how the chronic inflammatory milieu of adipose tissue in obesity prevents thermogenic compensation for overnutrition.

  View details for DOI 10.1172/JCI144888

  View details for Web of Science ID 000651200300004

  View details for PubMedID 33571167

  View details for PubMedCentralID PMC8011904

• AIDA and UCP1 snuggle up to prevent hypothermia NATURE CELL BIOLOGY Mills, E. L., Xiao, H., Chouchani, E. T. 2021; 23 (3): 216-218

  View details for DOI 10.1038/s41556-021-00648-3

  View details for Web of Science ID 000626809600001

  View details for PubMedID 33723427

• pH-Gated Succinate Secretion Regulates Muscle Remodeling in Response to Exercise. Cell Reddy, A., Bozi, L. H., Yaghi, O. K., Mills, E. L., Xiao, H., Nicholson, H. E., Paschini, M., Paulo, J. A., Garrity, R., Laznik-Bogoslavski, D., Ferreira, J. C., Carl, C. S., Sjøberg, K. A., Wojtaszewski, J. F., Jeppesen, J. F., Kiens, B., Gygi, S. P., Richter, E. A., Mathis, D., Chouchani, E. T. 2020; 183 (1): 62-75.e17

  Abstract

  In response to skeletal muscle contraction during exercise, paracrine factors coordinate tissue remodeling, which underlies this healthy adaptation. Here we describe a pH-sensing metabolite signal that initiates muscle remodeling upon exercise. In mice and humans, exercising skeletal muscle releases the mitochondrial metabolite succinate into the local interstitium and circulation. Selective secretion of succinate is facilitated by its transient protonation, which occurs upon muscle cell acidification. In the protonated monocarboxylic form, succinate is rendered a transport substrate for monocarboxylate transporter 1, which facilitates pH-gated release. Upon secretion, succinate signals via its cognate receptor SUCNR1 in non-myofibrillar cells in muscle tissue to control muscle-remodeling transcriptional programs. This succinate-SUCNR1 signaling is required for paracrine regulation of muscle innervation, muscle matrix remodeling, and muscle strength in response to exercise training. In sum, we define a bioenergetic sensor in muscle that utilizes intracellular pH and succinate to coordinate tissue adaptation to exercise.

  View details for DOI 10.1016/j.cell.2020.08.039

  View details for PubMedID 32946811

  View details for PubMedCentralID PMC7778787

• Proteomics illuminates fat as key tissue in aging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Long, J. Z. 2020; 117 (19): 10111–12

  View details for DOI 10.1073/pnas.1919410117

  View details for Web of Science ID 000532837500004

• Sample multiplexing for targeted pathway proteomics in aging mice. Proceedings of the National Academy of Sciences of the United States of America Yu, Q., Xiao, H., Jedrychowski, M. P., Schweppe, D. K., Navarrete-Perea, J., Knott, J., Rogers, J., Chouchani, E. T., Gygi, S. P. 2020; 117 (18): 9723-9732

  Abstract

  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.

  View details for DOI 10.1073/pnas.1919410117

  View details for PubMedID 32332170

  View details for PubMedCentralID PMC7211924

• Systematic quantification of the dynamics of newly synthesized proteins unveiling their degradation pathways in human cells. Chemical science Tong, M., Smeekens, J. M., Xiao, H., Wu, R. 2020; 11 (13): 3557-3568

  Abstract

  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.

  View details for DOI 10.1039/c9sc06479f

  View details for PubMedID 34109028

  View details for PubMedCentralID PMC8152571

• A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging. Cell Xiao, H., Jedrychowski, M. P., Schweppe, D. K., Huttlin, E. L., Yu, Q., Heppner, D. E., Li, J., Long, J., Mills, E. L., Szpyt, J., He, Z., Du, G., Garrity, R., Reddy, A., Vaites, L. P., Paulo, J. A., Zhang, T., Gray, N. S., Gygi, S. P., Chouchani, E. T. 2020; 180 (5): 968-983.e24

  Abstract

  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.

  View details for DOI 10.1016/j.cell.2020.02.012

  View details for PubMedID 32109415

  View details for PubMedCentralID PMC8164166

• Comprehensive Analysis of Protein Glycation Reveals Its Potential Impacts on Protein Degradation and Gene Expression in Human Cells. Journal of the American Society for Mass Spectrometry Sun, F., Suttapitugsakul, S., Xiao, H., Wu, R. 2019; 30 (12): 2480-2490

  Abstract

  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.

  View details for DOI 10.1007/s13361-019-02197-4

  View details for PubMedID 31073893

  View details for PubMedCentralID PMC6842084

• Global and site-specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry. Mass spectrometry reviews Xiao, H., Sun, F., Suttapitugsakul, S., Wu, R. 2019; 38 (4-5): 356-379

  Abstract

  Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site-specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)-based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS-based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS-based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX-XX, 2019.

  View details for DOI 10.1002/mas.21586

  View details for PubMedID 30605224

  View details for PubMedCentralID PMC6610820

• Author Correction: Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion. Nature communications Nguyen, D. C., Garimalla, S., Xiao, H., Kyu, S., Albizua, I., Galipeau, J., Chiang, K. Y., Waller, E. K., Wu, R., Gibson, G., Roberson, J., Lund, F. E., Randall, T. D., Sanz, I., Lee, F. E. 2019; 10 (1): 372

  Abstract

  The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'A patent has been applied for by Emory University with F.E.L, I.S. and D.C. N. as named inventors. The patent application number is PCT/US2016/036650'. This has now been corrected in both the PDF and HTML versions of the Article.

  View details for DOI 10.1038/s41467-019-08400-0

  View details for PubMedID 30655540

  View details for PubMedCentralID PMC6336838

• Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion. Nature communications Nguyen, D. C., Garimalla, S., Xiao, H., Kyu, S., Albizua, I., Galipeau, J., Chiang, K. Y., Waller, E. K., Wu, R., Gibson, G., Roberson, J., Lund, F. E., Randall, T. D., Sanz, I., Lee, F. E. 2018; 9 (1): 3698

  Abstract

  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.

  View details for DOI 10.1038/s41467-018-05853-7

  View details for PubMedID 30209264

  View details for PubMedCentralID PMC6135805

• Mass Spectrometry-Based Chemical and Enzymatic Methods for Global Analysis of Protein Glycosylation. Accounts of chemical research Xiao, H., Suttapitugsakul, S., Sun, F., Wu, R. 2018; 51 (8): 1796-1806

  Abstract

  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.

  View details for DOI 10.1021/acs.accounts.8b00200

  View details for PubMedID 30011186

  View details for PubMedCentralID PMC6118346

• Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods. International journal of mass spectrometry Xiao, H., Hwang, J. E., Wu, R. 2018; 429: 66-75

  Abstract

  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.

  View details for DOI 10.1016/j.ijms.2017.05.010

  View details for PubMedID 30147434

  View details for PubMedCentralID PMC6103449

• An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins. Nature communications Xiao, H., Chen, W., Smeekens, J. M., Wu, R. 2018; 9 (1): 1692

  Abstract

  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.

  View details for DOI 10.1038/s41467-018-04081-3

  View details for PubMedID 29703890

  View details for PubMedCentralID PMC5923262

• Extracellular vesicles from bone marrow-derived mesenchymal stromal cells support ex vivo survival of human antibody secreting cells. Journal of extracellular vesicles Nguyen, D. C., Lewis, H. C., Joyner, C., Warren, V., Xiao, H., Kissick, H. T., Wu, R., Galipeau, J., Lee, F. E. 2018; 7 (1): 1463778

  Abstract

  Extracellular vesicles (EVs) from bone marrow (BM)-derived mesenchymal stromal cells (BM-MSC) are novel mechanisms of cell-cell communication over short and long distances. BM-MSC have been shown to support human antibody secreting cells (ASC) survival ex vivo, but whether the crosstalk between the MSC-ASC interaction can occur via EVs is not known. Thus, we evaluated the role of EVs in ASC survival and IgG secretion. EVs were isolated from irradiated and non-irradiated primary BM-MSC and were quantified. They were further characterized by electron microscopy (EM) and CD63 and CD81 immuno-gold EM staining. Human ASC were isolated via fluorescence-activated cell sorting (FACS) and cultured ex vivo with the EV fractions, the EV-reduced fractions, or conventional media. IgG Elispots were used to measure the survival and functionality of the ASC. Contents of the EV fractions were evaluated by proteomics. We saw that both irradiated and non-irradiated MSC secretome preparations afforded vesicles of a size consistent with EVs. Both preparations appeared comparable in EM morphology and CD63 and CD81 immuno-gold EM. Both irradiated and non-irradiated EV fractions supported ASC function, at 88% and 90%, respectively, by day 3. In contrast, conventional media maintained only 4% ASC survival by day 3. To identify the specific factors that provided in vitro ASC support, we compared proteomes of the irradiated and non-irradiated EV fractions with conventional media. Pathway analysis of these proteins identified factors involved in the vesicle-mediated delivery of integrin signalling proteins. These findings indicate that BM-MSC EVs provide an effective support system for ASC survival and IgG secretion.

  View details for DOI 10.1080/20013078.2018.1463778

  View details for PubMedID 29713426

  View details for PubMedCentralID PMC5917896

• Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics. Molecular bioSystems Suttapitugsakul, S., Xiao, H., Smeekens, J., Wu, R. 2017; 13 (12): 2574-2582

  Abstract

  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.

  View details for DOI 10.1039/c7mb00393e

  View details for PubMedID 29019370

  View details for PubMedCentralID PMC5698164

• Simultaneous Quantitation of Glycoprotein Degradation and Synthesis Rates by Integrating Isotope Labeling, Chemical Enrichment, and Multiplexed Proteomics. Analytical chemistry Xiao, H., Wu, R. 2017; 89 (19): 10361-10367

  Abstract

  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.

  View details for DOI 10.1021/acs.analchem.7b02241

  View details for PubMedID 28850217

  View details for PubMedCentralID PMC5678942

• Evidence for the importance of post-transcriptional regulatory changes in ovarian cancer progression and the contribution of miRNAs. Scientific reports Zhang, M., Matyunina, L. V., Walker, L. D., Chen, W., Xiao, H., Benigno, B. B., Wu, R., McDonald, J. F. 2017; 7 (1): 8171

  Abstract

  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.

  View details for DOI 10.1038/s41598-017-08502-z

  View details for PubMedID 28811560

  View details for PubMedCentralID PMC5557889

• Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ali, M. K., Wu, Y., Tang, Y., Xiao, H., Chen, K., Han, T., Fang, N., Wu, R., El-Sayed, M. A. 2017; 114 (28): E5655-E5663

  Abstract

  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.

  View details for DOI 10.1073/pnas.1703151114

  View details for Web of Science ID 000405177100022

  View details for PubMedID 28652358

  View details for PubMedCentralID PMC5514737

• Specific Identification of Glycoproteins Bearing the Tn Antigen in Human Cells. Angewandte Chemie (International ed. in English) Zheng, J., Xiao, H., Wu, R. 2017; 56 (25): 7107-7111

  Abstract

  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.

  View details for DOI 10.1002/anie.201702191

  View details for PubMedID 28514044

  View details for PubMedCentralID PMC5529048

• Efficacy, long-term toxicity, and mechanistic studies of gold nanorods photothermal therapy of cancer in xenograft mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ali, M. K., Rahman, M., Wu, Y., Han, T., Peng, X., Mackey, M. A., Wang, D., Shin, H., Chen, Z. G., Xiao, H., Wu, R., Tang, Y., Shin, D. M., El-Sayed, M. A. 2017; 114 (15): E3110-E3118

  Abstract

  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.

  View details for DOI 10.1073/pnas.1619302114

  View details for Web of Science ID 000398789800016

  View details for PubMedID 28356516

  View details for PubMedCentralID PMC5393247

• Global and Site-Specific Analysis Revealing Unexpected and Extensive Protein S-GlcNAcylation in Human Cells. Analytical chemistry Xiao, H., Wu, R. 2017; 89 (6): 3656-3663

  Abstract

  Protein glycosylation is highly diverse and essential for mammalian cell survival. Heterogeneous glycans may be bound to different amino acid residues, forming multiple types of protein glycosylation. In this work, unexpected protein S-GlcNAcylation on cysteine residues was observed to extensively exist in human cells through global and site-specific analysis of protein GlcNAcylation by mass spectrometry. Three independent experiments produced similar results of many cysteine residues bound to N-acetylglucosamine (GlcNAc). Among well-localized S-GlcNAcylation sites, several motifs with an acidic amino acid around the sites were identified, which strongly suggests that a particular type of enzyme is responsible for this modification. Clustering results show that glycoproteins modified with S-GlcNAc are mainly involved in cell-cell adhesion and gene expression. For the first time, we found that proteins were extensively bound to GlcNAc through the side chains of cysteine residues in human cells, and the current discovery further advances our understanding of protein glycosylation.

  View details for DOI 10.1021/acs.analchem.6b05064

  View details for PubMedID 28234450

• Global Analysis of Secreted Proteins and Glycoproteins in Saccharomyces cerevisiae. Journal of proteome research Smeekens, J. M., Xiao, H., Wu, R. 2017; 16 (2): 1039-1049

  Abstract

  Protein secretion is essential for numerous cellular activities, and secreted proteins in bodily fluids are a promising and noninvasive source of biomarkers for disease detection. Systematic analysis of secreted proteins and glycoproteins will provide insight into protein function and cellular activities. Yeast (Saccharomyces cerevisiae) is an excellent model system for eukaryotic cells, but global analysis of secreted proteins and glycoproteins in yeast is challenging due to the low abundances of secreted proteins and contamination from high-abundance intracellular proteins. Here, by using mild separation of secreted proteins from cells, we comprehensively identified and quantified secreted proteins and glycoproteins through inhibition of glycosylation and mass spectrometry-based proteomics. In biological triplicate experiments, 245 secreted proteins were identified, and comparison with previous experimental and computational results demonstrated that many identified proteins were located in the extracellular space. Most quantified secreted proteins were down-regulated from cells treated with an N-glycosylation inhibitor (tunicamycin). The quantitative results strongly suggest that the secretion of these down-regulated proteins was regulated by glycosylation, while the secretion of proteins with minimal abundance changes was contrarily irrelevant to protein glycosylation, likely being secreted through nonclassical pathways. Glycoproteins in the yeast secretome were globally analyzed for the first time. A total of 27 proteins were quantified in at least two protein and glycosylation triplicate experiments, and all except one were down-regulated under N-glycosylation inhibition, which is solid experimental evidence to further demonstrate that the secretion of these proteins is regulated by their glycosylation. These results provide valuable insight into protein secretion, which will further advance protein secretion and disease studies.

  View details for DOI 10.1021/acs.jproteome.6b00953

  View details for PubMedID 27933904

• Quantitative investigation of human cell surface N-glycoprotein dynamics. Chemical science Xiao, H., Wu, R. 2017; 8 (1): 268-277

  Abstract

  Surface glycoproteins regulate nearly every extracellular event and they are dynamic for cells to adapt to the ever-changing extracellular environment. These glycoproteins contain a wealth of information on cellular development and disease states, and have significant biomedical implications. Systematic investigation of surface glycoproteins will result in a better understanding of surface protein functions, cellular activities and the molecular mechanisms of disease. However, it is extraordinarily challenging to specifically and globally analyze surface glycoproteins. Here we designed the first method to systematically analyze surface glycoprotein dynamics and measure their half-lives by integrating pulse-chase labeling, selective enrichment of surface glycoproteins, and multiplexed proteomics. The current results clearly demonstrated that surface glycoproteins with catalytic activities were more stable than those with binding and receptor activities. Glycosylation sites located outside of any domain had a notably longer median half-life than those within domains, which strongly suggests that glycans within domains regulate protein interactions with other molecules while those outside of domains mainly play a role in protecting the protein from degradation. This method can be extensively applied to biological and biomedical research.

  View details for DOI 10.1039/c6sc01814a

  View details for PubMedID 28616130

  View details for PubMedCentralID PMC5458730

• Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Ali, M. K., Wu, Y., Hang, T., Zang, X., Xiao, H., Tang, Y., Wu, R., Fernandez, F. M., El-Sayed, M. A. 2016; 138 (47): 15434-15442

  Abstract

  In cancer plasmonic photothermal therapy (PPTT), plasmonic nanoparticles are used to convert light into localized heat, leading to cancer cell death. Among plasmonic nanoparticles, gold nanorods (AuNRs) with specific dimensions enabling them to absorb near-infrared laser light have been widely used. The detailed mechanism of PPTT therapy, however, still remains poorly understood. Typically, surface-enhanced Raman spectroscopy (SERS) has been used to detect time-dependent changes in the intensity of the vibration frequencies of molecules that appear or disappear during different cellular processes. A complete proven assignment of the molecular identity of these vibrations and their biological importance has not yet been accomplished. Mass spectrometry (MS) is a powerful technique that is able to accurately identify molecules in chemical mixtures by observing their m/z values and fragmentation patterns. Here, we complemented the study of changes in SERS spectra with MS-based metabolomics and proteomics to identify the chemical species responsible for the observed changes in SERS band intensities during PPTT. We observed an increase in intensity of the bands at around 1000, 1207, and 1580 cm-1, which were assigned in the literature to phenylalanine, albeit with dispute. Our metabolomics results showed increased levels of phenylalanine, its derivatives, and phenylalanine-containing peptides, providing evidence for more confidence in the SERS peak assignments. To better understand the mechanism of phenylalanine increase upon PPTT, we combined metabolomics and proteomics results through network analysis, which proved that phenylalanine metabolism was perturbed. Furthermore, several apoptosis pathways were activated via key proteins (e.g., HADHA and ACAT1), consistent with the proposed role of altered phenylalanine metabolism in inducing apoptosis. Our study shows that the integration of the SERS with MS-based metabolomics and proteomics can assist the assignment of signals in SERS spectra and further characterize the related molecular mechanisms of the cellular processes involved in PPTT.

  View details for DOI 10.1021/jacs.6b08787

  View details for Web of Science ID 000389160500021

  View details for PubMedID 27809520

• Quantification of tunicamycin-induced protein expression and N-glycosylation changes in yeast. The Analyst Xiao, H., Smeekens, J. M., Wu, R. 2016; 141 (12): 3737-45

  Abstract

  Tunicamycin is a potent protein N-glycosylation inhibitor that has frequently been used to manipulate protein glycosylation in cells. However, protein expression and glycosylation changes as a result of tunicamycin treatment are still unclear. Using yeast as a model system, we systematically investigated the cellular response to tunicamycin at the proteome and N-glycoproteome levels. By utilizing modern mass spectrometry-based proteomics, we quantified 4259 proteins, which nearly covers the entire yeast proteome. After the three-hour tunicamycin treatment, more than 5% of proteins were down-regulated by at least 2 fold, among which proteins related to several glycan metabolism and glycolysis-related pathways were highly enriched. Furthermore, several proteins in the canonical unfolded protein response pathway were up-regulated because the inhibition of protein N-glycosylation impacts protein folding and trafficking. We also comprehensively quantified protein glycosylation changes in tunicamycin-treated cells, and more than one third of quantified unique glycopeptides (168 of 465 peptides) were down-regulated. Proteins containing down-regulated glycopeptides were related to glycosylation, glycoprotein metabolic processes, carbohydrate processes, and cell wall organization according to gene ontology clustering. The current results provide the first global view of the cellular response to tunicamycin at the proteome and glycoproteome levels.

  View details for DOI 10.1039/c6an00144k

  View details for PubMedID 27007503

• Site-Specific Quantification of Surface N-Glycoproteins in Statin-Treated Liver Cells. Analytical chemistry Xiao, H., Tang, G. X., Wu, R. 2016; 88 (6): 3324-32

  Abstract

  The frequent modification of cell-surface proteins by N-linked glycans is known to be correlated with many biological processes. Aberrant glycosylation on surface proteins is associated with different cellular statuses and disease progression. However, it is extraordinarily challenging to comprehensively and site-specifically analyze glycoproteins located only on the cell surface. Currently mass spectrometry (MS)-based proteomics provides the possibility to analyze the N-glycoproteome, but effective separation and enrichment methods are required for the analysis of surface glycoproteins prior to MS measurement. The introduction of bio-orthogonal groups into proteins accelerates research in the robust visualization, identification, and quantification of proteins. Here we have comprehensively evaluated different sugar analogs in the analysis of cell-surface N-glycoproteins by combining copper-free click chemistry and MS-based proteomics. Comparison of three sugar analogs, N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz), and N-azidoacetylmannosamine (ManNAz), showed that metabolic labeling with GalNAz resulted in the greatest number of glycoproteins and glycosylation sites in biological duplicate experiments. GalNAz was then employed for the quantification experiment in statin-treated HepG2 liver cells, and 280 unique N-glycosylated sites were quantified from 168 surface proteins. The quantification results demonstrated that many glycosylation sites on surface proteins were down-regulated in statin-treated cells compared to untreated cells because statin prevents the synthesis of dolichol, which is essential for the formation of dolichol-linked precursor oligosaccharides. Several glycosylation sites in proteins that participate in the Alzheimer's disease pathway were down-regulated. This method can be extensively applied for the global analysis of the cell-surface N-glycoproteome.

  View details for DOI 10.1021/acs.analchem.5b04871

  View details for PubMedID 26894747

• Systematic investigation of cellular response and pleiotropic effects in atorvastatin-treated liver cells by MS-based proteomics. Journal of proteome research Xiao, H., Chen, W., Tang, G. X., Smeekens, J. M., Wu, R. 2015; 14 (3): 1600-11

  Abstract

  For decades, statins have been widely used to lower cholesterol levels by inhibiting the enzyme HMG Co-A reductase (HMGCR). It is well-known that statins have pleiotropic effects including improving endothelial function and inhibiting vascular inflammation and oxidation. However, the cellular responses to statins and corresponding pleiotropic effects are largely unknown at the proteome level. Emerging mass spectrometry-based proteomics provides a unique opportunity to systemically investigate protein and phosphoprotein abundance changes as a result of statin treatment. Many lipid-related protein abundances were increased in HepG2 cells treated by atorvastatin, including HMGCR, FDFT, SQLE, and LDLR, while the abundances of proteins involved in cellular response to stress and apoptosis were decreased. Comprehensive analysis of protein phosphorylation demonstrated that several basic motifs were enriched among down-regulated phosphorylation sites, which indicates that kinases with preference for these motifs, such as protein kinase A and protein kinase C, have attenuated activities. Phosphopeptides on a group of G-protein modulators were up-regulated, which strongly suggests that cell signal rewiring was a result of the effect of protein lipidation by the statin. This work provides a global view of liver cell responses to atorvastatin at the proteome and phosphoproteome levels, which provides insight into the pleiotropic effects of statins.

  View details for DOI 10.1021/pr501277g

  View details for PubMedID 25668447