Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling.

The mechanistic target of rapamycin-mLST8-raptor complex (mTORC1) functions as a central regulator of cell growth and metabolism in response to changes in nutrient signals such as amino acids. SAMTOR is an S-adenosylmethionine (SAM) sensor, which regulates the mTORC1 activity through its interaction with the GTPase-activating protein activity toward Rags-1 (GATOR1)-KPTN, ITFG2, C12orf66 and SZT2-containing regulator (KICSTOR) complex. In this work, we report the crystal structures of Drosophila melanogaster SAMTOR in apo form and in complex with SAM. SAMTOR comprises an N-terminal helical domain and a C-terminal SAM-dependent methyltransferase (MTase) domain. The MTase domain contains the SAM-binding site and the potential GATOR1-KICSTOR-binding site. The helical domain functions as a molecular switch, which undergoes conformational change upon SAM binding and thereby modulates the interaction of SAMTOR with GATOR1-KICSTOR. The functional roles of the key residues and the helical domain are validated by functional assays. Our structural and functional data together reveal the molecular mechanism of the SAM sensing of SAMTOR and its functional role in mTORC1 signaling.

Radiographic measurement of the posterior tibial slope in normal Chinese adults: a retrospective cohort study.

BACKGROUND: Measurement of the posterior tibial slope (PTS) angle has important applications in total knee replacement surgery, high tibial osteotomy, and anterior cruciate ligament reconstruction. This study aimed to determine the mean PTS of knee joints in healthy Chinese adults, and provide data to guide knee surgery in China. METHODS: A retrospective analysis of 1257 (n = 1233, 50.4% male) plain X-ray films of participants aged 25-59 years was performed. The picture archiving and communication system was used for PTS measurement. The PTS was defined as the angle between the vertical line of the tangent of the anterior tibial cortex of the proximal tibia, and the tangent line of the tibial cortex. Two imaging physicians conducted the PTS measurements independently, and both the inter- and intraclass correlation coefficients (ICCs) were calculated. RESULTS: The mean PTS value was 7.68 ± 3.84° (range: 0-21°). The left PTS was significantly smaller in males than in females (7.22 ± 3.89 vs 8.05 ± 3.60; P = 0.005). Additionally, the PTS in participants aged 25-29 years was significantly larger than that in the other age groups (Left side: 8.64 ± 3.73 vs 6.92 ± 3.42, 7.42 ± 3.75, 7.53 ± 3.98; P <  0.001 and Right side: 8.68 ± 3.84 vs 7.48 ± 4.21, 7.13 ± 3.64, 7.66 ± 3.80; P = 0.004). There were no significant differences in PTS between the left and right sides. Two-way analysis of variance suggested that the differences in PTS between age groups were not affected by sex. The interobserver ICC was 0.91 (95% confidence interval [CI]: 0.85-0.94), and the intraobserver ICC was 0.90 (95% CI: 0.82-0.94). CONCLUSIONS: This study demonstrated that there were significant differences in PTS based on sex and age, highlighting the need to provide individualized treatment for knee surgery. It provided valuable information regarding the normal PTS values in Chinese adults and presented regionalised data to guide knee surgery.

Identification and Tetramer Structure of Hemin-Binding Protein SPD_0310 Linked to Iron Homeostasis and Virulence of Streptococcus pneumoniae.

Iron and iron-containing compounds are essential for bacterial virulence and host infection. Hemin is an important supplement compound for bacterial survival in an iron-deficient environment. Despite strong interest in hemin metabolism, the detailed mechanism of hemin transportation in Gram-positive bacteria is yet to be reported. The results of our study revealed that the homologous proteins of SPD_0310 were significantly conservative in Gram-positive bacteria (P < 0.001), and these proteins were identified as belonging to an uncharacterized protein family (UPF0371). The results of thermodynamic and kinetic studies have shown that SPD_0310 has a high hemin-binding affinity. Interestingly, we found that the crystal structure of SPD_0310 presented a homotetramer conformation, which is required for hemin binding. SPD_0310 can interact with many hemin-binding proteins (SPD_0090, SPD_1609, and GAPDH) located on the cell surface, which contributes to hemin transfer to the cytoplasm. It also has a high affinity with other iron transporters in the cytoplasm (SPD_0226 and SPD_0227), which facilitates iron redistribution in cells. More importantly, the knockout of the spd_0310 gene (Δspd_0310) resulted in a decrease in the iron content and protein expression levels of many bacterial adhesion factors. Moreover, the animal model showed that the Δspd_0310 strain has a lower virulence than the wild type. Based on the crystallographic and biochemical studies, we inferred that SPD_0310 is a hemin intermediate transporter which contributes to iron homeostasis and further affects the virulence of Streptococcus pneumoniae in the host. Our study provides not only an important theoretical basis for the in-depth elucidation of the hemin transport mechanism in bacteria but also an important candidate target for the development of novel antimicrobial agents based on metal transport systems. IMPORTANCE Iron is an essential element for bacterial virulence and infection of the host. The detailed hemin metabolism in Gram-positive bacteria has rarely been studied. SPD_0310 belongs to the UPF0371 family of proteins, and results of homology analysis and evolutionary tree analysis suggested that it was widely distributed and highly conserved in Gram-positive bacteria. However, the function of the UPF0371 family remains unknown. We successfully determined the crystal structure of apo-SPD_0310, which is a homotetramer. We found that cytoplasmic protein SPD_0310 with a special tetramer structure has a strong hemin-binding ability and interacts with many iron transporters, which facilitates hemin transfer from the extracellular space to the cytoplasm. The results of detailed functional analyses indicated that SPD_0310 may function as a hemin transporter similar to hemoglobin in animals and contributes to bacterial iron homeostasis and virulence. This study provides a novel target for the development of antimicrobial drugs against pathogenic Gram-positive bacteria.

AKT2 reduces IFNß1 production to modulate antiviral responses and systemic lupus erythematosus.

Interferon regulatory factor 3 (IRF3)-induced type I interferon (I-IFN) production plays key roles in both antiviral and autoimmune responses. IRF3 phosphorylation, dimerization, and nuclear localization are needed for its activation and function, but the precise regulatory mechanisms remain to be explored. Here, we show that the serine/threonine kinase AKT2 interacts with IRF3 and phosphorylates it on Thr207, thereby attenuating IRF3 nuclear translocation in a 14-3-3ε-dependent manner and reducing I-IFN production. We further find that AKT2 expression is downregulated in viral-infected macrophages or in monocytes and tissue samples from systemic lupus erythematosus (SLE) patients and mouse models. Akt2-deficient mice exhibit increased I-IFN induction and reduced mortality in response to viral infection, but aggravated severity of SLE. Overexpression of AKT2 kinase-inactive or IRF3-T207A mutants in zebrafish supports that AKT2 negatively regulates I-IFN production and antiviral response in a kinase-dependent manner. This negative role of AKT2 in IRF3-induced I-IFN production suggests that AKT2 may be therapeutically targeted to differentially regulate antiviral infection and SLE.

Nde1 is a Rab9 effector for loading late endosomes to cytoplasmic dynein motor complex.

Rab9 is mainly located on late endosomes and required for their intracellular transport to trans-Golgi network (TGN). The cytoplasmic dynein motor, together with its regulatory proteins Nde1/Ndel1 and Lis1, controls intracellular retrograde transport of membranous organelles along the microtubule network. How late endosomes are tethered to the microtubule-based motor dynein for their retrograde transport remains unclear. Here, we demonstrate that the guanosine triphosphate (GTP)-bound Rab9A/B specifically uses Nde1/Ndel1 as an effector to interact with the dynein motor complex. We determined the crystal structure of Rab9A-GTP in complex with the Rab9-binding region of Nde1. The functional roles of key residues involved in the Rab9A-Nde1 interaction are verified using biochemical and cell biology assays. Rab9A mutants unable to bind to Nde1 also failed to associate with dynein, Lis1, and dynactin. Therefore, Nde1 is a Rab9 effector that tethers Rab9-associated late endosomes to the dynein motor for their retrograde transport to the TGN.

HBO1 is a versatile histone acyltransferase critical for promoter histone acylations.

Recent studies demonstrate that histones are subjected to a series of short-chain fatty acid modifications that is known as histone acylations. However, the enzymes responsible for histone acylations in vivo are not well characterized. Here, we report that HBO1 is a versatile histone acyltransferase that catalyzes not only histone acetylation but also propionylation, butyrylation and crotonylation both in vivo and in vitro and does so in a JADE or BRPF family scaffold protein-dependent manner. We show that the minimal HBO1/BRPF2 complex can accommodate acetyl-CoA, propionyl-CoA, butyryl-CoA and crotonyl-CoA. Comparison of CBP and HBO1 reveals that they catalyze histone acylations at overlapping as well as distinct sites, with HBO1 being the key enzyme for H3K14 acylations. Genome-wide chromatin immunoprecipitation assay demonstrates that HBO1 is highly enriched at and contributes to bulk histone acylations on the transcriptional start sites of active transcribed genes. HBO1 promoter intensity highly correlates with the level of promoter histone acylation, but has no significant correlation with level of transcription. We also show that HBO1 is associated with a subset of DNA replication origins. Collectively our study establishes HBO1 as a versatile histone acyltransferase that links histone acylations to promoter acylations and selection of DNA replication origins.

MRI study of changes in knee bone marrow edema-like signal in asymptomatic amateur marathon runners before and after half-marathon running.

OBJECTIVE: To evaluate the incidence of knee bone marrow edema-like signal and its changes before and after running a half marathon running in asymptomatic amateur marathon runners to explore the impact of the half marathon on knee bone marrow edema-like signal. METHODS: 50 asymptomatic amateur marathon runners (30 males, 20 females) were recruited. T1-weighted imaging (T1WI), fat-suppressed protein density weighted imaging (fs-PDWI) and three-dimensional double-echo steady-state (3D-DESS) sequence on the right knee were performed before and within 3 h after a half-marathon running. 20 healthy volunteers were recruited as control. According to the whole-organ magnetic resonance imaging score (WORMS) system, the involvement of bone marrow edema-like signal in 15 regions of knee was graded from 0 to 3. The results were classified and Mann Whitney U test was used for comparison between groups. RESULTS: The total incidence of bone marrow edema-like signal in amateur marathon group was 62%. Among them, the incidence of grade 1-3 was 48% (24/50), 12% (6/50), 2% (1/50), respectively, which was statistically significant compared with the controls (P = 0.007). There was no significant difference between gender before running (P = 0.172) and after running (P = 0.162). There was no significant difference before and after running (P > 0.05). However, 3 subjects showed new lesions, 8 subjects showed progression and 4 subjects showed decreased signal. CONCLUSION: The occurrence of knee bone marrow edema-like signal in amateur marathon runners is more common. The lesions of bone marrow edema-like signal will show aggravation or improvement in a certain extent after the half marathon.

Structure, substrate specificity, and catalytic mechanism of human D-2-HGDH and insights into pathogenicity of disease-associated mutations.

D-2-hydroxyglutarate dehydrogenase (D-2-HGDH) catalyzes the oxidation of D-2-hydroxyglutarate (D-2-HG) into 2-oxoglutarate, and genetic D-2-HGDH deficiency leads to abnormal accumulation of D-2-HG which causes type I D-2-hydroxyglutaric aciduria and is associated with diffuse large B-cell lymphoma. This work reports the crystal structures of human D-2-HGDH in apo form and in complexes with D-2-HG, D-malate, D-lactate, L-2-HG, and 2-oxoglutarate, respectively. D-2-HGDH comprises a FAD-binding domain, a substrate-binding domain, and a small C-terminal domain. The active site is located at the interface of the FAD-binding domain and the substrate-binding domain. The functional roles of the key residues involved in the substrate binding and catalytic reaction and the mutations identified in D-2-HGDH-deficient diseases are analyzed by biochemical studies. The structural and biochemical data together reveal the molecular mechanism of the substrate specificity and catalytic reaction of D-2-HGDH and provide insights into the pathogenicity of the disease-associated mutations.

Imaging manifestations and pathological analysis of intramuscular myxoma: A case report and literature review.

Intramuscular Myxoma(IM) is a rare benign soft tissue tumor, and its etiology and histology source is still unclear. It is important to understand the pathological components of IM and its corresponding imaging features, as well as performing accurate and careful imaging assessments of IM before surgery. We present a case of a 43-year-old male who presented a lump in his left thigh and gradually enlarged during the past 8 years. The patient underwent CT, MRI, and CTA examined and was later pathologically confirmed as IM. This article will combine the literature，to explore the imaging manifestations and its pathological basis of intramuscular myxoma.

MRI features of and factors related to ankle injuries in asymptomatic amateur marathon runners.

OBJECTIVE: To analyze the MRI manifestations of and factors related to ankle injuries in asymptomatic amateur marathon runners. MATERIALS AND METHODS: A total of 113 amateur marathon runners without any ankle joint symptoms were recruited. Each participant was asked to complete a questionnaire at the beginning of the study and underwent MRI of the ankle. The MRI manifestations of ankle injuries were summarized, and binary logistic regression analysis was applied to analyze the factors related to ankle injuries. RESULTS: The main MRI features were bone marrow edema-like signal intensity, peritendinous effusion, and partial lateral collateral ligament injury. Others included Achilles tendinopathy, cyst-like lesions, osteochondral lesions, and subcutaneous soft tissue edema. The risk factor for bone marrow edema-like signal intensity in amateur marathon runners was a rearfoot strike pattern (p = 0.028, OR = 1.172); the risk factors for peritendinous effusion were a higher weekly running distance (p = 0.013, OR = 1.685) and increased running years (p = 0.039, OR = 1.113), whereas a rearfoot strike pattern (p = 0.005, OR = 0.831) was a protective factor for peritendinous effusion; the risk factor for Achilles tendinopathy was increased age (p = 0.008, OR = 1.412); the risk factors for anterior talofibular ligament injury were a rearfoot strike pattern (p = 0.017, OR = 1.346) and higher weekly running distance (p = 0.022, OR = 1.171); and the factors for calcaneofibular ligament injury were a higher weekly running distance (p = 0.029, OR = 1.570) and rearfoot strike pattern (p = 0.035, OR = 1.463). CONCLUSION: The main MRI features of asymptomatic amateur marathon runners are bone marrow edema-like signal intensity, peritendinous effusion, and partial lateral collateral ligament injury. In addition, increased age, increased running years, higher weekly running distance, and different foot strike patterns are risk factors for ankle injuries.

Structure basis of the FERM domain of kindlin-3 in supporting integrin αIIbß3 activation in platelets.

Kindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain-containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbß3 signaling in platelets by binding to the integrin ß3 cytoplasmic tail. However, the structural details of kindlin-3's FERM domain remain unknown. In this study, we crystalized the kindlin-3's FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3's FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbß3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbß3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbß3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbß3 activation.

Molecular mechanism of the dual regulatory roles of ATP on the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase.

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α2ßγ heterotetramer composed of the αß and αγ heterodimers. Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.

Probable risk factors of internal jugular vein stenosis in Chinese patients-A real-world cohort study.

OBJECTIVES: Extracranial venous anomalies, especially internal jugular vein stenosis (IJVS), have recently received increasing attention, however, its etiologies are uncertain. This study aimed to explore the probable risk factors of IJVS in Chinese PATIENTS AND METHODS: Eligible patients with IJVS confirmed by contrast-enhanced magnetic resonance venography (CE-MRV) were enrolled from October 2017 through October 2018. Probable risk factors were analyzed, including the conditions that may result in IJV wall damage, extraluminal compression, gender and age. RESULTS: A total of 133 patients enrolled in the final analysis, including 73 females and 60 males, the mean age were 54.83 ±â€¯15.25 years. In this IJVS cohort, the top two risks were previous hepatitis B virus (HBV) infection (48.9 %) and osseous compression (41.4 %). The IJVS cohort was divided into two subsets: extraluminal compression and non-compression. In the former, osseous compression (80.9 %) was the top risk factor, other risks including arterial (22.1 %) and lymph node compression (2.9 %). While, in the latter subset, the most common risk factor was previous HBV infection (46.2 %). In addition, cerebral venous sinus thrombosis (CVST) in non-compression subset was more common than that in extraluminal compression subset (21.5 % VS. 2.9 %, p = 0.001). When considered the gender (Male vs. Female), the ratios were 28.3 % vs. 0 % of smoking, p < 0.001, 16.67 % vs. 1.37 % of hyperhomocysteinemia, p = 0.002, and 11.67 % vs. 1.37 % of hyperuricemia, p = 0.023. In the subset with age less than 45 years, the top three risks included CVST (56.25 %), immunological diseases (55.56 %), and hyperhomocysteinemia (50.00 %), while, in the subset with the ages over 60 years, type-2 diabetes (66.66 %), carotid artery compression (53.33 %), previous HBV infection (52.31 %), and osseous compression (49.09 %) were more common than others. CONCLUSION: This study illustrates the probable risks of IJVS may be diverse, in which osseous compression and previous HBV infection may be the top two probable risks of IJVS in Chinese. This is the biggest difference from previous reports based on Caucasian.

Aldolase B suppresses hepatocellular carcinogenesis by inhibiting G6PD and pentose phosphate pathways.

Metabolic reprogramming is a core hallmark of cancer but it remains poorly defined in hepatocellular carcinogenesis (HCC). Here we show that hepatic aldolase B (Aldob) suppresses HCC by directly binding and inhibiting the rate-limiting enzyme in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD). A stage-dependent decrease of Aldob and increase of G6PD in human tumors are correlated with poor prognosis for patients with HCC. Global or liver-specific Aldob knockout promotes tumorigenesis in mice through enhancing G6PD activity and pentose phosphate pathway metabolism, whereas pharmacological inhibition or genetic knockdown of G6PD suppresses HCC. Consistently, restoration of Aldob in Aldob knockout mice attenuates tumorigenesis. We further demonstrate that Aldob potentiates p53-mediated inhibition of G6PD in an Aldob-G6PD-p53 complex. This scaffolding effect is independent of Aldob enzymatic activity. Together, our study reveals a new mode of metabolic reprogramming in HCC due to the loss of Aldob, suggesting a potential therapeutic strategy for HCC treatment.

Comparing hepatic steatosis distribution patterns between non-alcoholic fatty liver disease and fatty liver disease with chronic hepatitis B by second-harmonic generation/two-photon excited fluorescence method.

INTRODUCTION AND OBJECTIVES: Hepatitis B virus (HBV) might be an etiological factor modulating fat distribution in steatotic livers. We aim to compare hepatic steatosis distribution patterns between NAFLD and FL&CHB patients with second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) method. PATIENTS AND METHODS: 42 patients with NAFLD, 46 with FL&CHB and 55 without steatosis were enrolled in the study. Overall and regional steatosis in liver sections were quantified by SHG/TPEF method. The accuracy of which was validated by pathologist evaluation and magnetic resonance spectroscopy (MRS). Difference in degree of overall and regional steatosis between NAFLD and FL&CHB groups was analyzed by Mann-Whitney U test. Multivariable linear regression analysis was used to model factors contributing to steatosis distribution. RESULTS: The hepatic steatosis measured by SHG/TPEF method was highly correlated with pathologist grading (r=0.83, p<0.001) and MRS measurement (r=0.82, p<0.001). The level of overall steatosis in FL&CHB group is significantly lower than that in NAFLD group (p<0.001). In NAFLD group, periportal region has significantly lower steatosis percentage than lobule region and overall region (p<0.001); while in FL&CHB group there is no difference among regions. The ratio of steatosis at periportal region to lobule region is significantly higher in FL&CHB group than that in NAFLD group (p<0.05). Multivariable linear regression analysis shows that HBV infection is the major contributing factor (ß=0.322, p<0.01). CONCLUSIONS: SHG/TPEF method is an accurate and objective method in hepatic steatosis quantification. By quantifying steatosis in different histological regions, we found steatosis distribution patterns are different between FL&CHB and NAFLD patients.

Brain metastases from lung cancer with neuropsychiatric symptoms as the first symptoms.

BACKGROUND: To investigate the neuropsychiatric symptoms and their treatment and outcomes in lung cancer patients with brain metastases (BM), with an attempt to achieve early detection and prompt management of these symptoms. METHODS: Ten lung cancer patients (8 males and 2 females) with BMs who were treated in our center from 2013 to 2019 were enrolled in this analysis. Without exception, all 10 patients presented with chief complaints of neuropsychiatric symptoms, and BMs were eventually diagnosed. Appropriate treatments were offered, and all patients were followed up. RESULTS: Two patients died (case 5 died of sudden massive hemoptysis, and case 6 died after his families refused to receive the invasive treatment). Data on 3- and 5-year survival have been obtained from one patient each. The average follow-up duration was 19.4 months (except that two patients were hospitalized only once, and one patient received the second follow-up visit only 9 days after the first visit). CONCLUSIONS: The possibility of BM from lung cancer should be considered when a lung cancer patient develops neuropsychiatric symptoms, and timely diagnosis treatment should be arranged accordingly.

Role of Rab5 in the formation of macrophage-derived foam cell.

BACKGROUND: Foam cells play a key role in the occurrence and pathogenesis of atherosclerosis. Its formation starts with the ingestion of oxidized low-density lipoprotein (oxLDL). The process is associated with Ras related protein in brain 5 (Rab5) which plays a critical role in regulating endocytosis and early endosomal trafficking. Base on this, we presumed that Rab5 might participate in the maturation of foam cell. The aim of this study is to investigate the effect of Rab5 on macrophage cholesterol during the evolvement of macrophage when induced by oxLDL to the formation of foam cell. METHODS: Immunohistochemistry was performed to analyze the distribution of macrophages and Rab5 in atherosclerotic plaque. RNA inteference study and transfection of inactive mutant (GFP-Rab5-S34N) and active mutant (GFP-Rab5-Q79L) in U937-derived macrophage were utilized to investigate the impact of Rab5 on the process of macrophage cholesterol, which could be detected by oil red O staining, determination of intracellular lipid content, filipin staining, nile red staining and the costaining of early endosome antigen-1 (EEA-1) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylin dicarbocyanine (Dil)-labelled oxLDL (Dil-oxLDL). RESULTS: Rab5 was found abundantly localized in macrophage rich areas of human atherosclerotic lesions. On the foam cell study, the expression of Rab5 was increased after the incubation of oxLDL. The inteference study indicated the depletion of Rab5 led to the decreases of oil red O staining areas, total cholesterol and cholesterol esters in U937-derived marophages. Moreover, the fluorescence intensity of filipin and nile red staining were lower in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. The confocal study demonstrated less Dil-oxLDL was internalized in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L; the result showed also the decrease in colocalization of internalized Dil-oxLDL and EEA-1 for GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. CONCLUSIONS: Rab5 plays an important role in modulating the intracellular cholesterol of macrophages and consequently mediating the formation of foam cells.

Molecular basis for the recognition of CCN1 by monoclonal antibody 093G9.

CCN1, also named Cyr61 (cysteine-rich protein 61), is the first identified member of the CCN family that is composed of 6 secreted extracellular matrix-associated glycoproteins. CCN1 has been demonstrated to participate in pathogenesis of rheumatoid arthritis through various pathways. A monoclonal antibody, namely, 093G9, is effective to antagonize the effects of CCN1 and hence has potential therapeutic benefits against rheumatoid arthritis. Here, we show that the epitope recognized by 093G9 is mapped to residues 77 to 80 of CCN1, and a cyclic peptide encompassing residues 75 to 81 of CCN1 displays high binding affinity for 093G9. The crystal structure of the 093G9 Fab in complex with the cyclic peptide was determined at 2.7 Å resolution, which reveals the intensive interactions between CCN1 and 093G9. Particularly, residues Asn79 and Phe80 of CCN1 are inserted into cavities mainly formed by residues of complementarity-determining region loop L3 and framework region L2 and by residues of complementarity-determining region loops H2 and H3, respectively, which contribute most of the interactions and therefore are critical for the recognition by 093G9. Together, these findings not only identify the epitope of CCN1 for 093G9 but also reveal the molecular mechanism of recognition and binding of CCN1 by 093G9.

Structural and mechanistic insights into regulation of HBO1 histone acetyltransferase activity by BRPF2.

HBO1, a member of the MYST family of histone acetyltransferases (HATs), is required for global acetylation of histone H3K14 and embryonic development. It functions as a catalytic subunit in multisubunit complexes comprising a BRPF1/2/3 or JADE1/2/3 scaffold protein, and two accessory proteins. BRPF2 has been shown to be important for the HAT activity of HBO1 toward H3K14. Here we demonstrated that BRPF2 can regulate the HAT activity of HBO1 toward free H3 and H4, and nucleosomal H3. Particularly, a short N-terminal region of BRPF2 is sufficient for binding to HBO1 and can potentiate its activity toward H3K14. The crystal structure of the HBO1 MYST domain in complex with this segment of BRPF2 together with the biochemical and cell biological data revealed the key residues responsible for the HBO1-BRPF2 interaction. Our structural and functional data together indicate that the N-terminal region of BRPF2 plays an important role in the binding of HBO1 and a minor role in the binding of nucleosomes, which provide new mechanistic insights into the regulation of the HAT activity of HBO1 by BRPF2.

The ß and γ subunits play distinct functional roles in the α2ßγ heterotetramer of human NAD-dependent isocitrate dehydrogenase.

Human NAD-dependent isocitrate dehydrogenase existing as the α2ßγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory ß and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αß and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αß and αγ alone have considerable basal activity but the full activity of α2ßγ requires the assembly and cooperative function of both heterodimers. α2ßγ and αγ can be activated by citrate or/and ADP, whereas αß cannot. The binding of citrate or/and ADP decreases the S0.5,isocitrate and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate α2ßγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αß. Furthermore, the allosteric activation of α2ßγ is through the γ subunit not the ß subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the ß subunit the structural role to facilitate the assembly of the holoenzyme.