Sehyun Chae

A protein profile of visceral adipose tissues linked to early pathogenesis of type 2 diabetes mellitus

Adipose tissue is increasingly recognized as an endocrine organ playing important pathophysiological roles in metabolic abnormalities, such as obesity, cardiovascular disease, and type 2 diabetes mellitus (T2DM). In particular, visceral adipose tissue (VAT), as opposed to subcutaneous adipose tissue, is closely linked to the pathogenesis of insulin resistance and T2DM. Despite the importance of VAT, its molecular signatures related to the pathogenesis of T2DM have not been systematically explored. Here, we present comprehensive proteomic analysis of VATs in drug-naïve early T2DM patients and subjects with normal glucose tolerance. A total of 4,707 proteins were identified in LC-MS/MS experiments. Among them, 444 increased in abundance in T2DM and 328 decreased. They are involved in T2DM-related processes including inflammatory responses, peroxisome proliferator-activated receptor signaling, oxidative phosphorylation, fatty acid oxidation, and glucose metabolism. Of these proteins, we selected 11 VAT proteins that can represent alteration in early T2DM patients. Among them, up-regulation of FABP4, C1QA, S100A8, and SORBS1 and down-regulation of ACADL and PLIN4 were confirmed in VAT samples of independent early T2DM patients using Western blot. In summary, our profiling provided a comprehensive basis for understanding the link of a protein profile of VAT to early pathogenesis of T2DM.

Identification of Novel Autoantibodies in Type 1 Diabetic Patients Using a High-Density Protein Microarray

Autoantibodies can facilitate diagnostic and therapeutic means for type 1 diabetes (T1DM). We profiled autoantibodies from serum samples of 16 T1DM patients, 16 type 2 diabetic (T2DM) patients, and 27 healthy control subjects with normal glucose tolerance (NGT) by using protein microarrays containing 9,480 proteins. Two novel autoantibodies, anti-EEF1A1 and anti-UBE2L3, were selected from microarrays followed by immunofluorescence staining of pancreas. We then tested the validity of the candidates by ELISA in two independent test cohorts: 1) 95 adults with T1DM, 49 with T2DM, 11 with latent autoimmune diabetes in adults (LADA), 20 with Graves disease, and 66 with NGT and 2) 33 children with T1DM and 34 healthy children. Concentrations of these autoantibodies were significantly higher in T1DM patients than in NGT and T2DM subjects (P < 0.01), which was also confirmed in the test cohort of children (P < 0.05). Prevalence of anti-EEF1A1 and anti-UBE2L3 antibodies was 29.5% and 35.8% in T1DM, respectively. Of note, 40.9% of T1DM patients who lack anti-GAD antibodies (GADA) had anti-EEF1A1 and/or anti-UBE2L3 antibodies. These were also detected in patients with fulminant T1DM but not LADA. Our approach identified autoantibodies that can provide a new dimension of information indicative of T1DM independent of GADA and new insights into diagnosis and classification of T1DM.

Patterns of gene and metabolite define the effects of extracellular osmolality on kidney collecting duct.

To investigate the effects of changes in extracellular osmolality on the function of kidney collecting duct cells, particularly on water and sodium reabsorption in the conditions of diuresis and antidiuresis, we generated transcriptome and metabolome profiles of primary cultured inner medullary collecting duct (IMCD) cells. They were grown in hyperosmolar culture medium (640 mOsm) for 4 days and then exposed to either reduced (300 mOsm) or same osmolality for 1 or 2 days more. Integrated analysis of the transcriptome and metabolome revealed that decreased extracellular osmolality was associated with decreased levels of organic osmolytes, glucose, intermediates of citric acid cycle, and branched-chain amino acids (BCAA) in IMCD cells, along with significantly decreased gene expression and protein abundance of P-type transporters (ATP1B1), ABC transporters (ABCC5 and ABCG1), and insulin signaling pathways (IRS2). Quantitative real-time RT-PCR and semiquantitative immunoblotting confirmed the changes of transcript levels of differentially expressed genes and protein levels. Taken together, integrated analysis of omics data demonstrated that water and sodium reabsorption could be reduced by decreased extracellular osmolality per se, through decreased levels of ABC transporters and IRS2, which play a potential role in the transport of organic osmolytes, BCAA, glucose, and trafficking of epithelial sodium channel.

SUMO-Specific Protease 2 (SENP2) Is an Important Regulator of Fatty Acid Metabolism in Skeletal Muscle

Small ubiquitin-like modifier (SUMO)-specific proteases (SENPs) that reverse protein modification by SUMO are involved in the control of numerous cellular processes, including transcription, cell division, and cancer development. However, the physiological function of SENPs in energy metabolism remains unclear. Here, we investigated the role of SENP2 in fatty acid metabolism in C2C12 myotubes and in vivo. In C2C12 myotubes, treatment with saturated fatty acids, like palmitate, led to nuclear factor-κB–mediated increase in the expression of SENP2. This increase promoted the recruitment of peroxisome proliferator–activated receptor (PPAR)δ and PPARγ, through desumoylation of PPARs, to the promoters of the genes involved in fatty acid oxidation (FAO), such as carnitine-palmitoyl transferase-1 (CPT1b) and long-chain acyl-CoA synthetase 1 (ACSL1). In addition, SENP2 overexpression substantially increased FAO in C2C12 myotubes. Consistent with the cell culture system, muscle-specific SENP2 overexpression led to a marked increase in the mRNA levels of CPT1b and ACSL1 and thereby in FAO in the skeletal muscle, which ultimately alleviated high-fat diet–induced obesity and insulin resistance. Collectively, these data identify SENP2 as an important regulator of fatty acid metabolism in skeletal muscle and further implicate that muscle SENP2 could be a novel therapeutic target for the treatment of obesity-linked metabolic disorders.

Compact variant‐rich customized sequence database and a fast and sensitive database search for efficient proteogenomic analyses

In proteogenomic analysis, construction of a compact, customized database from mRNA‐seq data and a sensitive search of both reference and customized databases are essential to accurately determine protein abundances and structural variations at the protein level. However, these tasks have not been systematically explored, but rather performed in an ad‐hoc fashion. Here, we present an effective method for constructing a compact database containing comprehensive sequences of sample‐specific variants—single nucleotide variants, insertions/deletions, and stop‐codon mutations derived from Exome‐seq and RNA‐seq data. It, however, occupies less space by storing variant peptides, not variant proteins. We also present an efficient search method for both customized and reference databases. The separate searches of the two databases increase the search time, and a unified search is less sensitive to identify variant peptides due to the smaller size of the customized database, compared to the reference database, in the target‐decoy setting. Our method searches the unified database once, but performs target‐decoy validations separately. Experimental results show that our approach is as fast as the unified search and as sensitive as the separate searches. Our customized database includes mutation information in the headers of variant peptides, thereby facilitating the inspection of peptide‐spectrum matches.

Comprehensive proteome profiling of platelet identified a protein profile predictive of responses to an antiplatelet agent sarpogrelate.

Sarpogrelate is an antiplatelet agent widely used to treat arterial occlusive diseases. Evaluation of platelet aggregation is essential to monitor therapeutic effects of sarpogrelate. Currently, no molecular signatures are available to evaluate platelet aggregation. Here, we performed comprehensive proteome profiling of platelets collected from 18 subjects before and after sarpogrelate administration using LC-MS/MS analysis coupled with extensive fractionation. Of 5423 proteins detected, we identified 499 proteins affected by sarpogrelate and found that they strongly represented cellular processes related to platelet activation and aggregation, including cell activation, coagulation, and vesicle-mediated transports. Based on the network model of the proteins involved in these processes, we selected three proteins (cut-like homeobox 1; coagulation factor XIII, B polypeptide; and peptidylprolyl isomerase D) that reflect the platelet aggregation-related processes after confirming their alterations by sarpogrelate in independent samples using Western blotting. Our proteomic approach provided a protein profile predictive of therapeutic effects of sarpogrelate.

Quantitative Proteomics Reveals β2 Integrin-mediated Cytoskeletal Rearrangement in Vascular Endothelial Growth Factor (VEGF)-induced Retinal Vascular Hyperpermeability

Retinal vascular hyperpermeability causes macular edema, leading to visual deterioration in retinal diseases such as diabetic retinopathy and retinal vascular occlusion. Dysregulation of junction integrity between endothelial cells by vascular endothelial growth factor (VEGF) was shown to cause retinal vascular hyperpermeability. Accordingly, anti-VEGF agents have been used to treat retinal vascular hyperpermeability. However, they can confer potential toxicity through their deleterious effects on maintenance and survival of neuronal and endothelial cells in the retina. Thus, it is important to identify novel therapeutic targets for retinal vascular hyperpermeability other than VEGF. Here, we prepared murine retinas showing VEGF-induced vascular leakage from superficial retinal vascular plexus and prevention of VEGF-induced leakage by anti-VEGF antibody treatment. We then performed comprehensive proteome profiling of these samples and identified retinal proteins for which abundances were differentially expressed by VEGF, but such alterations were inhibited by anti-VEGF antibody. Functional enrichment and network analyses of these proteins revealed the β2 integrin pathway, which can prevent dysregulation of junction integrity between endothelial cells through cytoskeletal rearrangement, as a potential therapeutic target for retinal vascular hyperpermeability. Finally, we experimentally demonstrated that inhibition of the β2 integrin pathway salvaged VEGF-induced retinal vascular hyperpermeability, supporting its validity as an alternative therapeutic target to anti-VEGF agents.

Prospective isolation of ISL1+ cardiac progenitors from human ESCs for myocardial infarction therapy

Summary The LIM-homeodomain transcription factor ISL1 marks multipotent cardiac progenitors that give rise to cardiac muscle, endothelium, and smooth muscle cells. ISL1+ progenitors can be derived from human pluripotent stem cells, but the inability to efficiently isolate pure populations has limited their characterization. Using a genetic selection strategy, we were able to highly enrich ISL1+ cells derived from human embryonic stem cells. Comparative quantitative proteomic analysis of enriched ISL1+ cells identified ALCAM (CD166) as a surface marker that enabled the isolation of ISL1+ progenitor cells. ALCAM+/ISL1+ progenitors are multipotent and differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Transplantation of ALCAM+ progenitors enhances tissue recovery, restores cardiac function, and improves angiogenesis through activation of AKT-MAPK signaling in a rat model of myocardial infarction, based on cardiac MRI and histology. Our study establishes an efficient method for scalable purification of human ISL1+ cardiac precursor cells for therapeutic applications.

Quantitative proteomic analysis of changes related to age and calorie restriction in rat liver tissue

Calorie restriction (CR) is the most frequently studied mechanism for increasing longevity, protecting against stress, and delaying age‐associated diseases. Most studies have initiated CR in young animals to determine the protective effects against aging. Although aging phenomena are well‐documented, the molecular mechanisms of aging and CR remain unclear. In this study, we observe changes in hepatic proteins upon age‐related and diet‐restricted changes in the rat liver using quantitative proteomics. Quantitative proteomes are measured using tandem mass tag labeling followed by LC‐MS/MS. We compare protein levels in livers from young (6 months old) and old (25 months old) rats with 40% calorie‐restricted (YCR and OCR, respectively) or ad libitum diets. In total, 44 279 peptides and 3134 proteins are identified and 260 differentially expressed proteins are found. Functional enrichment analysis show that these proteins are mainly involved in glucose and fatty acid metabolism‐related processes, consistent with the theory that energy metabolism regulation is dependent on age‐related and calorie‐restricted changes in liver tissue. In addition, proteins mediating inflammation and gluconeogenesis are increased in OCR livers, but not YCR livers. These results show that CR in old rats might not have antiaging benefits because liver inflammation is increased.

Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation

AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders.AMPK activation has been suggested as treatment for obesity and its complications. Here the authors show that the ubiquitin ligase MKRN1 binds to AMPK and mediates its ubiquitination and degradation. Loss of MKRN1 leads to AMPK activation, increased glucose consumption and decreased lipid accumulation.