Rheumatoid Arthritis Patient With Neurofibromatosis Type 1: Case Report and Review of the Literature.

A 66-year-old neurofibromatosis type 1 (NF1) patient with polyarticular pain for nine years, aggravated for two days, was transferred from the Emergency Intensive Care Unit (EICU) to our rheumatology department. She was diagnosed with NF1 nine years ago by a gene mutation detection and coronary heart disease (CHD) three months ago. The patient was diagnosed with rheumatoid arthritis (RA) this time. After 24 days of treatment with appropriate medication, the patient was discharged with relieved joint pain. However, about four months later, the patient died of circulatory failure caused by myocardial infarction. We analyzed the possible reasons for her outcome and made a review of the literature. There are few clinical reports of NF1 complicated with RA. We found five cases reported in the literature up to date during our search and included them in our communication to compare with our case. NF1 combined with RA mainly affects adult women and usually starts with NF1 and is followed by RA after at least six years of NF1 symptom onset. Although the summarized characteristics of clinical and potential pathogenesis of NF1 combined with RA were limited with these six cases, we hope that this will help clinicians to increase their understanding of this rare complication, thus helping to guide clinical medication.

Cryo-electron microscopy for GPCR research and drug discovery in endocrinology and metabolism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structures resolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5-7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, G protein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathway or an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.

Autoimmune diseases: targets, biology, and drug discovery.

Autoimmune diseases (AIDs) arise from a breakdown in immunological self-tolerance, wherein the adaptive immune system mistakenly attacks healthy cells, tissues and organs. AIDs impose excessive treatment costs and currently rely on non-specific and universal immunosuppression, which only offer symptomatic relief without addressing the underlying causes. AIDs are driven by autoantigens, targeting the autoantigens holds great promise in transforming the treatment of these diseases. To achieve this goal, a comprehensive understanding of the pathogenic mechanisms underlying different AIDs and the identification of specific autoantigens are critical. In this review, we categorize AIDs based on their underlying causes and compile information on autoantigens implicated in each disease, providing a roadmap for the development of novel immunotherapy regimens. We will focus on type 1 diabetes (T1D), which is an autoimmune disease characterized by irreversible destruction of insulin-producing ß cells in the Langerhans islets of the pancreas. We will discuss insulin as possible autoantigen of T1D and its role in T1D pathogenesis. Finally, we will review current treatments of TID and propose a potentially effective immunotherapy targeting autoantigens.

Role of the voltage-gated proton channel Hv1 in insulin secretion, glucose homeostasis, and obesity

Diabetes is characterized by an absolutely inadequate insulin secretion (type 1 diabetes mellitus) or a relative deficit in insulin secretion due to insulin resistance (type 2 diabetes mellitus), both of which result in elevated blood glucose. Understanding the molecular mechanisms underlying the pathophysiology of diabetes could lead to the development of new therapeutic approaches. The voltage-gated proton channel Hv1 is an ion channel with specific selectivity for protons, which is regulated by membrane potential and intracellular pH. Recently, our studies showed that Hv1 is expressed in β cells of the endocrine pancreas. Knockout of Hv1 reduces insulin secretion and results in hyperglycemia and glucose intolerance, but not insulin resistance. Furthermore, knockout of Hv1 leads to diet-induced obesity due to inflammation and hepatic steatosis. Increasing evidence suggests that Hv1 plays a pivotal role in glucose homeostasis and lipid metabolism. This review aims to summarize advances made so far in our understanding of the roles of Hv1 in the regulation of insulin secretion in β cells, glucose homeostasis, and obesity. (AU)

Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress.

Heat stress is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. Abnormally high environmental temperature induces oxidative stress and apoptosis in bovine mammary epithelial cells (BMECs). Nicotinamide mononucleotide (NMN) has beneficial effects in maintaining the cellular physiological functions. In this study, we evaluate the protective effect of NMN on heat stress-induced apoptosis of BMECs and explore the potential underlying mechanisms. Our results showed that heat stress considerably decreased cell viability in BMECs, whereas pretreatment of BMECs with NMN (150 µM) for 24 h significantly alleviated the negative effects of heat stress on cells. NMN protected BMECs from heat stress-induced oxidative stress by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited apoptosis by reducing the ratio of Bax/Bcl2 and blocking proteolytic the cleavage of Caspase-3 in heat stressed-BMECs. Importantly, NMN treatment could reduce mitochondrial damage through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (MFN1, 2); and suppress endoplasmic reticulum stress through unfolded protein response regulator Glucose regulated protein 78 (GRP78), and downstream elements Recombinant activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Above all, our results demonstrate that NMN supplemention attenuates heat stress-induced oxidative stress and apoptosis in BMECs by maintaining mitochondrial fission and fusion, and regulating endoplasmic reticulum stress, which provides the convincing evidence that NMN has valuable potential in alleviating mammary gland injury of dairy cows caused by environmental heat stress.

Role of the voltage-gated proton channel Hv1 in insulin secretion, glucose homeostasis, and obesity.

Diabetes is characterized by an absolutely inadequate insulin secretion (type 1 diabetes mellitus) or a relative deficit in insulin secretion due to insulin resistance (type 2 diabetes mellitus), both of which result in elevated blood glucose. Understanding the molecular mechanisms underlying the pathophysiology of diabetes could lead to the development of new therapeutic approaches. The voltage-gated proton channel Hv1 is an ion channel with specific selectivity for protons, which is regulated by membrane potential and intracellular pH. Recently, our studies showed that Hv1 is expressed in ß cells of the endocrine pancreas. Knockout of Hv1 reduces insulin secretion and results in hyperglycemia and glucose intolerance, but not insulin resistance. Furthermore, knockout of Hv1 leads to diet-induced obesity due to inflammation and hepatic steatosis. Increasing evidence suggests that Hv1 plays a pivotal role in glucose homeostasis and lipid metabolism. This review aims to summarize advances made so far in our understanding of the roles of Hv1 in the regulation of insulin secretion in ß cells, glucose homeostasis, and obesity.

Heparin interacts with the main protease of SARS-CoV-2 and inhibits its activity.

The ability of SARS-CoV-2 to replicate in host cells is dependent on its main protease (Mpro, also called 3CLpro) that cut the viral precursor polyproteins and is a major target for antiviral drug design. Here, we showed that heparin interacts with the Mpro of SARS-CoV-2 and inhibits its activity. Protein fluorescence quenching showed that heparin strongly binds to the Mpro protein with dissociation constants KD of 16.66 and 31.60 µM at 25 and 35 °C, respectively. From thermodynamic parameters of the interaction, there are hydrophobic and hydrogen bond interactions between them. Fluorescence resonance energy transfer (FRET) assay demonstrated that heparin inhibits the proteolytic activity of Mpro with an inhibition constant Ki of 6.9 nM and a half maximal inhibitory concentrations (IC50) of 7.8 ± 2.6 nM. Furthermore, molecular docking analysis revealed that the recognition and binding groups of heparin within the active site of SARS-CoV-2 Mpro provide important new information for the characteristics of the interactions of heparin with the protease. Our finding suggested that heparin might have a potential role in inhibiting SARS-CoV-2 infection through inhibiting Mpro activity of SARS-CoV-2.

Inhibition of porphyra polysaccharide on xanthine oxidase activity and its inhibition mechanism.

Xanthine oxidase (XO) is a purine catabolic enzyme related to hyperuricemia and gout. Porphyra polysaccharide (PP) is a kind of sulfated polysaccharide with potent biological activity. Herein, the interaction mechanism between PP and XO was studied by enzyme kinetics and multi-spectroscopy methods for the first time. Inhibition kinetics assay showed that PP reversibly inhibited XO activity in a mixed competitive manner with an IC50 of 10.53 ± 0.69 mg/ml. Fluorescence titration studies and thermodynamic parameter calculations revealed that PP could spontaneously bind to XO through hydrophobic interactions, with a class of binding site. Circular dichroism analysis demonstrated that PP induced secondary structure rearrangement and conformational change of XO. Molecular docking further revealed that PP inserted into the hydrophobic cavity of XO, occupying the catalytic center, leading to the inhibition of XO activity. This study may provide new insights into the inhibitory mechanism of PP as a promising XO inhibitor.

Mice lacking the proton channel Hv1 exhibit sex-specific differences in glucose homeostasis.

Sex as a physiologic factor has a strong association with the features of metabolic syndrome. Our previous study showed that loss of the voltage-gated proton channel Hv1 inhibits insulin secretion and leads to hyperglycemia and glucose intolerance in male mice. However, there are significant differences in blood glucose between male and female Hv1-knockout (KO) mice. Here, we investigated the differences in glucose metabolism and insulin sensitivity between male and female KO mice and how sex steroids contribute to these differences. We found that the fasting blood glucose in female KO mice was visibly lower than that in male KO mice, which was accompanied by hypotestosteronemia. KO mice in both sexes exhibited higher expression of gluconeogenesis-related genes in liver compared with WT mice. Also, the livers from KO males displayed a decrease in glycolysis-related gene expression and an increase in gluconeogenesis-related gene expression compared with KO females. Furthermore, exogenous testosterone supplementation decreased blood glucose levels in male KO mice, as well as enhancing insulin signaling. Taken together, our data demonstrate that knockout of Hv1 results in higher blood glucose levels in male than female mice, despite a decreased insulin secretion in both sexes. This sex-related difference in glucose homeostasis is associated with the glucose metabolism in liver tissue, likely due to the physiological levels of testosterone in KO male mice.

Inhibitory effects of chondroitin sulfate on alpha-amylase activity: A potential hypoglycemic agent.

Inhibiting the activity of the intestinal enzyme α-amylase that catalyzes the degradation of starch into glucose can control blood glucose and provide an essential way for the treatment of Type-II diabetes mellitus (T2DM). Here, we compared the structural information of chondroitin sulfate (CS) from different origins and the effects on activity of α-amylase and blood glucose have been investigated. The inhibitory effects of shark and porcine CSs against α-amylase activity is obvious with IC50 values of 11.97 and 14.42 mg/ml, respectively, but the bovine CS almost no effect. From the data of fluorescence spectroscopic analyses, CSs from shark and pig quench Try fluorescence intensity of the enzyme, whereas bovine CS induces an increase. In vivo, oral administration of shark and porcine CSs efficiently suppresses postprandial blood glucose levels in normal and diabetic mice. Our study found that CSs from different sources showed different biological functions even if both molecular weight and disaccharide subunit composition are almost the same, and demonstrated that the CSs from shark and pig as α-amylase inhibitors could be regarded as a novel functional food ingredient in T2DM management.

miRNA-10a-5p inhibits cell metastasis in hepatocellular carcinoma via targeting SKA1.

A variety of microRNAs (miRNAs) are involved in the occurrence and development of hepatocellular carcinoma (HCC). However, the role of miR-10a-5p in the progression of HCC remains unclear. Therefore, the purpose of this study was to determine the role of miR-10a-5p in the development of HCC and the possible molecular mechanism. miR-10a-5p expression in HCC tissues and plasma from patients was detected by quantitative real-time polymerase chain reaction. Migratory changes in HCC cells were detected after the overexpression of miR-10a-5p. Epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. Finally, through luciferase assay and rescue experiments, the mechanism by which miR-10a-5p regulates its downstream gene, human spindle and kinetochore-associated complex subunit 1, SKA1 and the interaction between these molecules in the development of HCC were determined. The expression of miR-10a-5p was markedly downregulated in HCC tissues, cell lines, and plasma. The overexpression of miR-10a-5p significantly inhibited the migration, invasion, and EMT of HCC cells. Furthermore, SKA1 was shown to be a downstream gene of miR-10a-5p. SKA1 silencing had the same effect as miR-10a-5p overexpression in HCC. In particular, the overexpression of SKA1 reversed the inhibitory effects of miR-10a-5p in HCC. Taken together, low miR-10a-5p expression is associated with HCC progression. miR-10a-5p inhibits the malignant development of HCC by negatively regulating SKA1.

Deficiency of voltage-gated proton channel Hv1 aggravates ovalbumin-induced allergic lung asthma in mice.

Asthma is a chronic airway inflammation that caused by many factors. The voltage-gated proton channel Hv1 has been proposed to extrude excessive protons produced by NADPH oxidase (NOX) from cytosol to maintain its activity during respiratory bursts. Here, we showed that loss of Hv1 aggravates ovalbumin (OVA)-induced allergic lung asthma in mice. The numbers of total cells, eosinophils and neutrophils in bronchoalveolar lavage fluid (BALF) of Hv1-deficiency (KO) mice are obviously increased after OVA challenge compared with that of wild-type (WT) mice. Histopathological staining reveals that Hv1-deficiency aggravates OVA-induced inflammatory cell infiltration and goblet cell hyperplasia in lung tissues. The expression of IL-4, IL-5 and IL-13 are markedly increased in lung tissues of OVA-challenged KO mice compared with that of WT mice. Furthermore, the expression levels of NOX2, NOX4 and DUOX1 are dramatically increased, while the expression levels of SOD2 and catalase are significantly reduced in lung tissues of OVA-challenged KO mice compared with that of WT mice. The production of ROS in lung tissues of KO mice is significantly higher than that of WT mice after OVA challenge. Our data suggest that Hv1-deficiency might aggravate the development of allergic asthma through increasing ROS production.

Loss of voltage-gated proton channel Hv1 leads to diet-induced obesity in mice.

OBJECTIVE: The voltage-gated proton channel Hv1 has been proposed to mediate NADPH oxidase (NOX) function by regulating intracellular pH during respiratory bursts. In our previous work, we showed that Hv1 is expressed in pancreatic ß cells and positively regulates insulin secretion. Here, we investigated the role of Hv1 in adipose tissue differentiation, metabolic homeostasis and insulin sensitivity using Hv1 knockout (KO) mice. DESIGN: Mice with genetic deletion of Hv1 are treated with high-fat diet (HFD) similar to wild-type (WT) mice. Body weight gain, adiposity, insulin sensitivity and gene expressions in both adipose tissue and liver were analyzed. RESULTS: Mice with genetic deletion of Hv1 display overt obesity with higher body weight gain and accumulation of adipose tissue compared with similarly HFD-treated WT. Hv1-deficient mice develop more glucose intolerance than WT, but no significant difference in insulin resistance, after fed with HFD. Deficiency of Hv1 results in a remarkable increase in epididymal adipocyte weight and size, while the gene expressions of proinflammatory factors and cytokines are obviously enhanced in the HFD-fed mice. Furthermore, the gene expression of Hv1 is increased in the HFD-fed mice, which is accompanied by the increase of NOX2 and NOX4. In addition, there is more severely diet-induced steatosis and inflammation in liver in KO mice. CONCLUSION: Our data demonstrated that lacking of Hv1 results in diet-induced obesity in mice through inflammation and hepatic steatosis. This study suggested that Hv1 acts as a positive regulator of metabolic homeostasis and a potential target for antiobesity drugs in therapy and may serve as an adaptive mechanism in cooperating with NOX to mediate reactive oxygen species for adipogenesis and insulin sensitivity.

Loss of the voltage-gated proton channel Hv1 decreases insulin secretion and leads to hyperglycemia and glucose intolerance in mice.

Insulin secretion by pancreatic islet ß-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic ß-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here we demonstrate that Hv1 promotes insulin secretion of pancreatic ß-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance because of reduced insulin secretion but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. Islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced the first and second phase of glucose-stimulated insulin secretion. Islet insulin and proinsulin content was reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of ß-cell mass in Hv1 knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity for glucose-induced membrane depolarization, accompanied by a reduced ability of glucose to raise Ca2+ levels in islets, as evidenced by decreased durations of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic ß-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with ß-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.

Hv1-deficiency protects ß cells from glucotoxicity through regulation of NOX4 level.

High glucose (HG)-induced oxidative stress contributes to the dysfunction of pancreatic ß cells in diabetes. The voltage-gated proton channel Hv1 has been proposed to support reactive oxygen species (ROS) production during respiratory bursts. However, the effect of Hv1 on glucotoxicity in pancreatic ß cells is not clear yet. In this study, we examined the protective effects of Hv1-deficiency in HG cultured ß cells. Following 48 h of treatment with 30 mM high glucose, Hv1 KO ß cells showed higher cell viability, lower cell apoptosis and a more stable insulin gene expression level compared to WT ß cells. In both control and HG cultured ß cells, deficiency of Hv1 decreased the glucose- and PMA-induced ROS production. Finally, HG incubation led to NOX4 upregulation in WT ß cells, which could be inhibited by HV1 deficiency. In conclusion, Hv1-deficiency prevents the HG treatment-induced NOX4 upregulation and protects ß cells from glucotoxicity.

Structural characterisation of a full-length mitochondrial outer membrane TOM40 preprotein translocase: implications for its interaction with presequence peptides.

Tom40, the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex), forms the import pore that facilitates the translocation of preproteins across the outer membrane. Though the function of Tom40 has been intensively studied, the details of the interactions between presequence peptides and Tom40 remain unclear. In this study, we expressed rat Tom40 in Escherichia coli and purified it from inclusion bodies before investigating the refolded protein by fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The far-UV CD spectra of the refolded Tom40 in various concentrations of urea revealed that the refolded protein has a well-defined structure consisting mainly of ß-sheet. Moreover, the specific binding of presequence peptides to Tom40, which was demonstrated by fluorescence quenching, showed that the refolded purified protein is functional and that the interaction between Tom40 and presequence peptides is mainly electrostatic in nature.

Deficiency of voltage-gated proton channel Hv1 attenuates streptozotocin-induced ß-cell damage.

Reactive oxygen species (ROS) impairs pancreatic ß-cells and plays an important role in development of diabetes. Streptozotocin (STZ) can lead to ß-cell dysfunction via inducing ROS production. The voltage-gated proton channel Hv1 contributes a majority of the charge compensation required for ROS production. Here, we investigated the effects of Hv1 on STZ-induced ß-cell damage. We found that deficiency of Hv1 obviously inhibits STZ-induced glucose intolerance in mice, and prevents the decrease in ß-cell mass and pancreatic insulin content from STZ-treatment. Further studies showed that loss of Hv1 significantly attenuates STZ-induced ß-cell damage and ROS production in pancreatic ß-cells. Our results suggest that Hv1 might contribute to development of diabetes through producing ROS.

Proton pump inhibitors have pH-dependent effects on the thermostability of the carboxyl-terminal domain of voltage-gated proton channel Hv1.

The voltage-gated proton channel Hv1 is highly selective for H+ and is activated by membrane depolarization and pH gradient. An increased external and decreased internal pH opens the Hv1 channel. The intracellular C-terminal domain of Hv1 is responsible for channel dimerization, cooperative, and thermosensitive gating. Here, we found that proton pump inhibitors (PPIs) interact with the C-terminal domain of human Hv1. The interaction between PPIs and the C-terminal domain, which is pH-dependent, lowered the thermal and structural stability of the protein at pH 4, but enhanced the thermal and structural stability at pH 8. Furthermore, we investigated in vitro the interaction of PPIs with the C-terminal domain of Hv1 by fluorescence and micro-Raman spectra. Fluorescence quenching measurements revealed that the interaction between the C-terminal domain and PPIs is a mainly hydrophobic interaction. The micro-Raman spectra showed that PPIs did not form stable disulfide bonds with the unique thiol group within this domain (Cys249 residue). The preferential interaction of PPIs with the inactive form of Hv1 stabilizes the high pH inactive state of the C-terminal domain, indicating a mechanism by which PPIs might act explicitly on the stabilization of a closed state of the proton channel.

Expression of soluble programmed death-1 protein in peripheral blood regulatory T cells and its effects on rheumatoid arthritis progression.

The present study aimed to investigate the role of the soluble programmed death­1 (sPD-1) protein, which is released by peripheral blood regulatory T cells (Treg) during the progression of rheumatoid arthritis (RA). From October 2012 to May 2014, 82 RA patients (RA group) and 90 healthy volunteers (healthy controls; HC) were recruited. Cluster of differentiation (CD)4, CD25 and forkhead/winged helix transcription factor p3 (Foxp3) and expression of cytotoxic T lymphocyte associated antigen 4 (CTLA-4) and Foxp3 were detected by flow cytometry. Expression of sPD­1 in Treg was detected by western blot analysis. Immunosuppressive activity of CD4+CD25­ Treg was measured via thiazolyl blue in an MTT assay. ELISA was used to detect interleukin­10 (IL­10), transforming growth factor beta (TGF-ß), interleukin-4 (IL-4), interferon­Î³ (IFN-γ) and nuclear factor of activated T cells (NF­AT). It was observed that in peripheral blood, CD4+CD25-FOXP3+/CD4+ levels were reduced in the RA group (P<0.001), and sPD­1 levels were markedly higher (P<0.001), compared with the HC group. Additionally, it was observed that relative sPD­1 protein expression in the small interfering RNA (siRNA)-sPD-1 treated group was reduced compared with the untreated and scrambled siRNA groups (all P<0.0001). The mean fluorescence intensity of CTLA-4 and Foxp3 decreased markedly upon transfection with siRNA-sPD-1 (P<0.001). Compared with the normal CD4+CD25­ T group, optical density (OD)540 values, IFN-γ/IL-4 concentration ratio and NF­AT activity in siRNA untreated and scramble groups reduced significantly (all P<0.001). OD540 value, IFN-γ/IL-4 concentration ratio and NF­AT activity in the siRNA­sPD­1 group were significantly upregulated (all P<0.001). Therefore, sPD-1 may suppress the level of CD4+CD25­ Tregs in the peripheral blood of RA patients, and may be involved in a variety of immune processes mediated by CD4+CD25­ Tregs.

Interaction of divalent metal ions with human translocase of inner membrane of mitochondria Tim23.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23IMS) and the interaction of presequence peptides with Tim23IMS in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23IMS. The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim23IMS is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex.