Characterization of peptide-fused protein assemblies in living cells.

Assembly of de novo peptides designed from scratch is in a semi-rational manner and creates artificial supramolecular structures with unique properties. Considering that the functions of various proteins in living cells are highly regulated by their assemblies, building artificial assemblies within cells holds the potential to simulate the functions of natural protein assemblies and engineer cellular activities for controlled manipulation. How can we evaluate the self-assembly of designed peptides in cells? The most effective approach involves the genetic fusion of fluorescent proteins (FPs). Expressing a self-assembling peptide fused with an FP within cells allows for evaluating assemblies through fluorescence signal. When µm-scale assemblies such as condensates are formed, the peptide assemblies can be directly observed by imaging. For sub-µm-scale assemblies, fluorescence correlation spectroscopy analysis is more practical. Additionally, the fluorescence resonance energy transfer (FRET) signal between FPs is valuable evidence of proximity. The decrease in fluorescence anisotropy associated with homo-FRET reveals the properties of self-assembly. Furthermore, by combining two FPs, one acting as a donor and the other as an acceptor, the heteromeric interaction between two different components can be studied through the FRET signal. In this chapter, we provide detailed protocols, from designing and constructing plasmid DNA expressing the peptide-fused protein to analysis of self-assembly in living cells.

Proximity labeling and identification of endogenous client proteins recruited to Y15-based artificial granules tethering a bait protein.

Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.

Downregulation of extramitochondrial BCKDH and its uncoupling from AMP deaminase in type 2 diabetic OLETF rat hearts.

Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

Role of Erythropoiesis-Stimulating Agents in Cardiovascular Protection in CKD Patients: Reappraisal of Their Impact and Mechanisms.

Erythropoiesis-stimulating agents (ESAs) have markedly reduced the need for blood transfusion for renal anemia and are included in standard therapies for patients with chronic kidney disease (CKD). Various protective effects of ESAs on the cardiovascular system have been discovered through basic research, and the effects have received much attention because the rates of cardiovascular events and mortality are high in CKD patients. However, randomized clinical trials did not provide strong evidence that ESAs exert cardioprotection in humans, including CKD patients. It is difficult to assess the cardioprotective effects of ESAs in CKD patients through the clinical data that has been reported to date because the relationship between hemoglobin level rather than ESA dose and cardiovascular event rates was examined in most studies. Interestingly, recent studies using a rat model of CKD showed that the infarct size-limiting effect of an ESA was lost when its dose was increased to a level that normalized blood hemoglobin levels, suggesting that the optimal dose of an ESA for myocardial protection is less than the dose required to normalize hemoglobin levels. Furthermore, animal models of traditional coronary risk factors or comorbidities were resistant to the cardioprotective effects of ESAs because of interruptions in signal-mediated mechanisms downstream of erythropoietin receptors. In this review, we briefly discuss basic and clinical data on the impact of anemia on coronary and systemic circulation, the effects of CKD on the cardiovascular system, and the multiple pharmacological actions of ESAs to examine whether the ESAs that are prescribed for renal anemia exert any cardioprotection in patients with CKD.

Pattern enrichment analysis for phage selection of stapled peptide ligands.

Phage display is the most widely used technique to discover de novo peptides that bind to target proteins. However, it is associated with some challenges such as compositional bias. In this study, to overcome these difficulties, we devised a 'pattern enrichment analysis.' In this method, two samples (one obtained by affinity selection, the other simply amplified without selection) are prepared, and the two sequence datasets read on next-generation sequencer are compared to find the three-residue pattern most enriched in the selected sample. This allows us to compare two sequence datasets with high coverage and facilitates the identification of peptide sequences and the key residues for binding. We also demonstrated that this approach in the combination with structured peptide libraries allowed spatial mapping of the enriched sequence patterns. Here, we prepared a phage library displaying chemically stapled helical peptides with the X1C2X3X4X5X6X7X8C9X10 sequence, where X is any amino acid. To validate our method, we performed screening against the HDM2 protein. The results showed that the hydrophobic residues (Phe, Tyr, Trp and Leu) that are key to interactions with HDM2 were clearly identified by the pattern enrichment analysis. We also performed selection targeting the SARS-CoV-2 spike RBD in the same manner. The results showed that similar patterns were enriched among the hit peptides that inhibited the protein-protein interaction.

Effects of Sacubitril/Valsartan on Glycemic Control in Japanese Patients With Heart Failure and/or Hypertension.

Background: Post hoc analysis of the PARADIGM-HF trial showed that sacubitril/valsartan (S/V) was more effective than enalapril in lowering HbA1c in patients with heart failure and diabetes. Methods and Results: In the present study, the effect of S/V on glycemic control was retrospectively analyzed in 150 patients (median age 74 years) who were prescribed S/V for the treatment of heart failure and/or hypertension. After a median period of 13 weeks treatment, mean (±SD) HbA1c levels decreased significantly from 6.56±0.68% to 6.49±0.63%. The decrease in HbA1c was evident in patients with (n=111), but not in those without, diabetes. There were no significant changes in renal function after S/V treatment, but systolic blood pressure was significantly reduced from 141±21 to 134±19 mmHg. Ninety patients had N-terminal pro B-type natriuretic peptide (NT-proBNP) tested, and S/V significantly decreased median NT-proBNP concentrations from 1,026 to 618 pg/mL; however, there was no correlation between the degree of decrease in HbA1c and that in NT-proBNP. Multiple regression analysis revealed that being diabetic, rather than having heart failure, was a significant independent variable for a reduction in HbA1c. Conclusions: Treatment with S/V improved glycemic control in patients with heart failure and/or hypertension, especially in those with concomitant diabetes. This favorable effect on glucose metabolism may be mediated by neprilysin inhibition and is desirable in the treatment of heart failure and hypertension in diabetic patients.

Effects of Hydrophobic Residues on the Intracellular Self-Assembly of De Novo Designed Peptide Tags and Their Orthogonality.

Protein assemblies forming nano- to micro-sized structures underlie versatile biological events in living systems. For mimicking and engineering these protein assemblies through a bottom-up approach, self-assembling peptides (SAPs) that form nanofibril structures via ß-sheets serve as potential practical tags. Nevertheless, the development of SAP tags is still in its infancy, and insight into the relationship between peptide sequences and intracellular self-assembly is limited. In this study, we focused on hydrophobic residues in SAPs and examined the self-assembly of SAP-tagged superfolder GFPs (green fluorescent proteins) in COS-7 cells. Based on XEXK (X; hydrophobic amino acids: F, L, I, V, W, or Y) sequence units, we designed a panel of Xn peptides with different hydrophobic residues (X) and chain lengths (n). We observed that the self-assembly propensity, the size of the assemblies, the influence on protein denaturation, and the subcellular localization differed significantly depending on the hydrophobic amino acid. F9, L9, I7, and V13 peptides formed µm-scaled granules, W13 formed small oligomeric clusters in the cytoplasm, and Y15 formed assemblies in the nucleus. In addition, we investigated the orthogonality of their interaction. Strikingly, W13- and Y15-tagged proteins interacted independently and formed two distinct assemblies in cells. Herein, we have demonstrated the great opportunities for rationalizing artificial protein assemblies and orthogonal structures in an intracellular context using the designed SAPs.

Impact of the COVID-19 Pandemic on Glycemic Control and Blood Pressure Control in Patients with Diabetes in Japan.

Objective In this study, we investigated whether and how the COVID-19 pandemic affected glycemic control and blood pressure (BP) control in patients with diabetes mellitus (DM). Methods DM patients whose HbA1c level was measured regularly before and after the declaration of a state of emergency were included in this study. Some patients were given questionnaires about changes in their lifestyle to determine the factors affecting glycemic control and BP control. Results The median HbA1c level of the 804 patients increased significantly from 6.8% before the state of emergency to 7.1% and 7.0% during and after the state of emergency, respectively. This was in contrast to the decrease one year earlier due to seasonal variations. In the 176 patients who responded to the questionnaire, the HbA1c level also increased significantly during and after the state of emergency. The worsening of glycemic control was more pronounced in the group that had achieved HbA1c of <7% before the state of emergency than in those with higher values. Unlike the rise in HbA1c, the BP did not rise during the state of emergency but did rise significantly afterwards. There was no marked decrease in HbA1c or BP after the state of emergency, even in patients who responded that they were much more careful with their diet, ate less, or exercised more. Conclusions The COVID-19 pandemic worsened glycemic control and BP control, even in patients who perceived no marked change in their diet or exercise, suggesting that more active lifestyle guidance is necessary for good treatment of DM patients.

A guide-tag system controlling client enrichment into Y15 peptide-based granules for an in-cell protein recruitment assay.

Self-assembling peptides (SAPs) are valuable building blocks for the fabrication of artificial supramolecules. We developed a guide-tag system that concentrates client proteins into SAP-based scaffolds in cellular environments at various enrichment levels. This system provides a tool to analyse the protein-protein interactions caused by protein clustering in cells.

Reduction in GLP-1 secretory capacity may be a novel independent risk factor of coronary artery stenosis.

Multiple factors regulate glucagon-like peptide-1 (GLP-1) secretion, but a group of apparently healthy subjects showed blunted responses of GLP-1 secretion in our previous study. In this study, we examined whether the reduction in GLP-1 secretory capacity is associated with increased extent of coronary artery stenosis in non-diabetic patients. Non-diabetic patients who were admitted for coronary angiography without a history of coronary interventions were enrolled. Coronary artery stenosis was quantified by Gensini score (GS), and GS ≥ 10 was used as an outcome variable based on its predictive value for cardiovascular events. The patients (mean age, 66.5 ± 8.8 years; 71% males, n = 173) underwent oral 75 g-glucose tolerant tests for determination of glucose, insulin and active GLP-1 levels. The area under the curve of plasma active GLP-1 (AUC-GLP-1) was determined as an index of GLP-1 secretory capacity. AUC-GLP-1 was not correlated with fasting glucose, AUC-glucose, serum lipids or indices of insulin sensitivity. In multivariate logistic regression analysis for GS ≥ 10, AUC-GLP-1 < median, age and hypertension were selected as explanatory variables, though fasting GLP-1 level was not selected. The findings suggest that reduction in GLP-1 secretory capacity is a novel independent risk factor of coronary stenosis.

Biofunctional supramolecular hydrogels fabricated from a short self-assembling peptide modified with bioactive sequences for the 3D culture of breast cancer MCF-7 cells.

Self-assembling peptides are a type of molecule with promise as scaffold materials for cancer cell engineering. We have reported a short self-assembling peptide, (FFiK)2, that had a symmetric structure connected via a urea bond. In this study, we functionalized (FFiK)2 by conjugation with various bioactive sequences for the 3D culture of cancer cells. Four sequences, RGDS and PHSRN derived from fibronectin and AG73 and C16 derived from laminin, were selected as bioactive sequences to promote cell adhesion, proliferation or migration. (FFiK)2, and its derivatives could co-assemble into supramolecular nanofibers displaying bioactive sequences and form hydrogels. MCF-7 cells were encapsulated in functionalized peptide hydrogels without significant cytotoxicity. Encapsulated MCF-7 cells proliferated under 3D culture conditions. MCF-7 cells proliferated with spheroid formation in hydrogels that displayed RGDS or PHSRN sequences, which will be able to be applied to drug screening targeting cancer stem cells. On the other hand, since MCF-7 cells migrated in a 3D hydrogel that displayed AG73, we could construct the metastatic model of breast cancer cells, which is helpful for the elucidation of breast cancer cells and drug screening against cancer cells under metastatic state. Therefore, functionalized (FFiK)2 hydrogels with various bioactive sequences can be used to regulate cancer cell function for tumor engineering and drug screening.

Intracellular artificial supramolecules based on de novo designed Y15 peptides.

De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms ß-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.

Characteristics of patients with emergency attendance for severe hypoglycemia and hyperglycemia in a general hospital in Japan.

ABSTRACT: Despite advances in treatments for diabetes mellitus (DM), severe acute glycemic crises still occur. In this study, the characteristics of patients who were transported to an emergency department due to acute glycemic crises were investigated.We enrolled patients who were transported to our hospital by ambulance due to hypoglycemia or hyperglycemia during the period from January 2015 to December 2019. Initial glucose levels below 70 mg/dL and above 250 mg/dL were defined as hypoglycemia and hyperglycemia, respectively.In the 5-year period, 16,910 patients were transported to our hospital by ambulance. Of those patients, 87 patients (0.51%) were diagnosed with hypoglycemia, 26 patients (0.15%) were diagnosed with hyperglycemia and 1 patient was diagnosed with lactic acidosis. Compared to patients with hypoglycemia, blood urea nitrogen, serum potassium and hemoglobin levels were higher in patients with hyperglycemia. Systolic blood pressure was lower and pulse rate was higher in patients with hyperglycemia, possibly reflecting dehydration in hyperglycemia. Patients with hyperglycemia were younger (63 vs 70 years old, median), more likely to be hospitalized (92.3% vs 23.0%) with poorer prognosis (23.1% vs 4.6%) than those with hypoglycemia. In 64 DM patients with hypoglycemia, 34 patients were treated with insulin and 24 patients were treated with sulfonylurea or glinide, and their medication was often inappropriate. Excessive alcohol intake and malnutrition were the main causes of hypoglycemia in 23 non-DM patients. The main reasons for hyperglycemia were interrupted treatment, forgetting insulin injection and infection.To avoid acute glycemic crises, optimization of anti-DM therapy and education of patients are needed.

Involvement of necroptosis in contrast-induced nephropathy in a rat CKD model.

BACKGROUND: The risk of contrast-induced nephropathy (CIN) is high in patients with chronic kidney disease (CKD). However, the mechanism of CIN in CKD is not fully understood. Here, we prepared a clinically relevant model of CIN and examined the role of necroptosis, which potentially cross-talks with autophagy, in CIN. METHODS: In Sprague-Dawley rats, CKD was induced by subtotal nephrectomy (SNx, 5/6 nephrectomy) 4 weeks before induction of CIN. CIN was induced by administration of a contrast medium (CM), iohexol, following administration of indomethacin and N-omega-Nitro-L-arginine methyl ester. Renal function and tissue injuries were assessed 48 h after CM injection. RESULTS: Serum creatinine (s-Cre) and BUN were increased from 0.28 ± 0.01 to 0.52 ± 0.02 mg/dl and from 15.1 ± 0.7 to 29.2 ± 1.2 mg/dl, respectively, after SNx alone. CM further increased s-Cre and BUN to 0.69 ± 0.03 and 37.2 ± 2.1, respectively. In the renal tissue after CM injection, protein levels of receptor-interacting serine/threonine-protein kinase (RIP) 1, RIP3, cleaved caspase 3, and caspase 8 were increased by 64 ~ 212%, while there was reduction in LC3-II and accumulation of p62. Necrostatin-1, an RIP1 inhibitor, administered before and 24 h after CM injection significantly suppressed elevation of s-Cre, BUN and urinary albumin levels, kidney injury molecule-1 expression and infiltration of CD68-positive macrophages in renal tissues after CM injection. CONCLUSION: The results suggest that necroptosis of proximal tubular cells contributes to CIN in CKD and that suppression of protective autophagy by pro-necroptotic signaling may also be involved.

Selection of fluorescent biosensors against galectin-3 from an NBD-modified phage library displaying designed α-helical peptides.

Fluorescent biosensors are indispensable tools for molecular imaging, detection, and drug screening. Conventionally, fluorescent biosensors were constructed by incorporating fluorophores into ligands. Here, to develop ligand-independent biosensors, we demonstrated biosensor selection from a fluorophore-modified peptide phage library. In this library, the peptides were designed to form α-helical structures, and one cysteine, the probe modification site, was located at the center of four randomized residues on the same face of the helix. By conjugation with 4-nitrobenzoxadiazole (NBD), we constructed an NBD-modified phage library. We conducted selection against galectin-3 (Gal-3), a galactose-specific lectin associated with various biological events such as tumor metastasis and insulin resistance. After biopanning, we obtained NBD-modified peptides that selectively bind to Gal-3 from the library. The fluorescence intensity of the hit biosensors increased with the concentration of Gal-3, and this fluorescent response was visually observed.

Xanthine oxidoreductase-mediated injury is amplified by upregulated AMP deaminase in type 2 diabetic rat hearts under the condition of pressure overload.

BACKGROUND: We previously reported that upregulated AMP deaminase (AMPD) contributes to diastolic ventricular dysfunction via depletion of the adenine nucleotide pool in a rat model of type 2 diabetes (T2DM), Otsuka Long-Evans-Tokushima Fatty rats (OLETF). Meanwhile, AMPD promotes the formation of substrates of xanthine oxidoreductase (XOR), which produces ROS as a byproduct. Here, we tested the hypothesis that a functional link between upregulated AMPD and XOR is involved in ventricular dysfunction in T2DM rats. METHODS AND RESULTS: Pressure-volume loop analysis revealed that pressure overloading by phenylephrine infusion induced severer left ventricular diastolic dysfunction (tau: 14.7 ± 0.8 vs 12.5 ± 0.7 msec, left ventricular end-diastolic pressure: 18.3 ± 1.5 vs 12.2 ± 1.3 mmHg, p < 0.05) and ventricular-arterial uncoupling in OLETF than in LETO, non-diabetic rats, though the baseline parameters were comparable in the two groups. While the pressure overload did not affect AMPD activity, it increased XOR activity both in OLETF and LETO, with OLETF showing significantly higher XOR activity than that in LETO (347.2 ± 17.9 vs 243.2 ± 6.1 µg/min/mg). Under the condition of pressure overload, myocardial ATP level was lower, and levels of xanthine and uric acid were higher in OLETF than in LETO. Addition of exogenous inosine, a product of AMP deamination, to the heart homogenates augmented XOR activity. OLETF showed 68% higher tissue ROS levels and 47% reduction in mitochondrial state 3 respiration compared with those in LETO. Overexpression of AMPD3 in H9c2 cells elevated levels of hypoxanthine and ROS and reduced the level of ATP. Inhibition of XOR suppressed the production of tissue ROS and mitochondrial dysfunction and improved ventricular function under the condition of pressure overload in OLETF. CONCLUSIONS: The results suggest that increases in the activity of XOR and the formation of XOR substrates by upregulated AMPD contribute to ROS-mediated diastolic ventricular dysfunction at the time of increased cardiac workload in diabetic hearts.

hDM2 protein-binding peptides screened from stapled α-helical peptide phage display libraries with different types of staple linkers.

Chemically modified peptide ligands were identified from α-helix peptide phage libraries with different types of staple linkers. The hDM2-protein was used as a representative target of protein-protein interactions to screen ligands for p53 binding sites in hDM2. Two types of staple linkers were used for the chemical modification of the peptide phage display libraries before affinity selection. The identified stapled peptides could bind to hDM2 competitively with the p53 peptide. The stapled peptide phage libraries developed in this study will improve the discovery of protein-protein interaction inhibitors through the synergistic effect of peptide units and staple linkers.

Distinct intra-mitochondrial localizations of pro-survival kinases and regulation of their functions by DUSP5 and PHLPP-1.

ERK and Akt have been shown to regulate cell sensitivity to death-inducing stress by phosphorylating GSK-3ß, a major modulator of the threshold for mitochondrial permeability transition. Here we examined intra-mitochondrial localization of the pro-survival kinases and their regulation by phosphatases. Stepwise trypsin digestion of mitochondria isolated from HEK293 or H9c2 cells was performed, and immunoblotting revealed that GSK-3ß and ERK localized dominantly in the outer membrane (OM), while Akt resided at comparable levels in OM, the inner membrane (IM) and the matrix. Treatment with IGF-1 increased the protein level of Akt in the matrix, while ERK and GSK-3ß protein levels were increased in OM. Simultaneously, IGF-1 treatment elevated the level of Thr202/Tyr204-phospho-ERK in IM and matrix and levels of Ser473-phospho-Akt and Ser9-phospho-GSK-3ß in OM, IM and matrix. Exposing cells to reactive oxygen species (ROS) by using antimycin A increased the levels of DUSP5 and PHLPP-1 mainly in OM and induced dephosphorylation of Akt, ERK and GSK-3ß. The mitochondrial localization of DUSP5 was confirmed by experiments with mitochondria purified by Percoll gradient centrifugation and by transfection of cells with GFP-tagged DUSP5. Knockdown of either DUSP5 or PHLPP-1 increased the levels of both Thr202/Tyr204-phospho-ERK and Ser473-phospho-Akt in mitochondria. Cell death induced by antimycin A was suppressed by siRNA-mediated knockdown of DUSP5. The results suggest that Akt and ERK in mitochondria show distinct intra-mitochondrial localization and crosstalk in GSK-3ß regulation and that recruitment of DUSP5 as well as PHLPP-1 to mitochondria contributes to ROS-induced termination of the protective signaling.

Empagliflozin attenuates acute kidney injury after myocardial infarction in diabetic rats.

Acute kidney injury (AKI) predicts poor prognosis in patients with acute myocardial infarction (MI) and diabetes mellitus (DM) is an independent risk factor of AKI. Recent clinical studies have shown the beneficial effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on cardiovascular and renal outcomes in patients with DM. We recently reported that canagliflozin normalized susceptibility of diabetic rats to AKI after acute MI via ß-hydroxybutyrate-mediated suppression of NOX expression. Here we examined whether the same renoprotective effect is shared by empagliflozin. Serum creatinine levels were not changed by MI induced by coronary artery occlusion in LETO, non-diabetic control rats, and OLETF, obese type 2 diabetic rats. However, immunohistochemistry revealed that MI increased renal expression of NGAL and KIM-1, early markers of tubular injury, by 3.2-fold and 2.6-fold, respectively, in OLETF. These increases in injury markers were not observed in LETO. Pretreatment with empagliflozin of OLETF for 2 weeks improved hyperglycemia, increased blood ß-hydroxybutyrate level, and suppressed MI-induced expression of NGAL and KIM-1. Empagliflozin suppressed upregulation of NOX2 and NOX4 in the kidney of OLETF. Taken together with the results of our previous study, it was concluded that treatment with the SGLT2 inhibitor protects the diabetic kidney from MI-induced AKI.

Construction of a Stapled α-Helix Peptide Library Displayed on Phage for the Screening of Galectin-3-Binding Peptide Ligands.

A stapled α-helix peptide library was designed and constructed using a chemically modified phage display system for screening stapled-peptide ligands against target proteins. The α-helix peptide library, with two cysteine residues on the opposite side of the randomized face, was modified with a rigid hydrocarbon staple linker on a phage. The stapled α-helix peptide phage library was screened against galectin-3 (Gal-3), a cancer-related galactose-binding protein. The obtained stapled peptides showed a high binding affinity (K d = 0.45 µM) despite being nonsugar ligands. The stapled modification played important roles in stabilizing the α-helical structure that contributed to the high binding affinity to Gal-3. In addition, the best stapled peptide ligands showed specific binding to Gal-3 among various carbohydrate-binding proteins. Thus, the designed α-helix peptide phage library with a constrained structure by the staple linker will advance the discovery of peptide ligands with improved specificity and affinity.