Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

Self-efficacy as a crucial psychological predictor of treatment adherence among elderly people living with HIV: analyses based on the health belief model.

Medication adherence to antiretroviral therapy (ART) among elderly people living with HIV (PLWH) is of serious concern. Our study aimed to understand the medication adherence of elderly PLWH under ART based on the health belief model (HBM). A baseline survey with a total of 529 elderly PLWH was conducted in Sichuan. Logistic and linear regression analysis, mediation analysis, and path analysis based on prior evidence were used. Only self-efficacy showed direct associations with medication adherence in the last four days (ORm = 1.37, 95%CI: 1.11, 1.70) and the last month (ORm = 1.39, 95%CI: 1.18, 1.63) in the multivariate analysis. Self-efficacy mediated the relations between perceived benefits, perceived barriers, cues to action and medication adherence. Inner relations existed within the HBM. In addition to the direct effects, perceived benefits (ß = 0.149, p = 0.031; ß = 0.093, p = 0.005), perceived barriers (ß = -0.070, p = 0.008; ß = -0.062, p = 0.012), and cues to action (ß = 0.184, p = 0.013; ß = 0.135, p = 0.014) showed indirect effects on medication adherence in the last four days and the last month, respectively. HBM may be effective in predicting medication adherence of elderly PLWH, and self-efficacy may be a crucial predictor and mediator. Efforts should be focused on how to enhance elderly PLWH's self-efficacy without neglect of other medication beliefs.

Association between social capital and mental health among older people living with HIV: the Sichuan Older HIV-Infected Cohort Study (SOHICS).

BACKGROUND: Mental health problems are common among older people living with HIV and associated with poorer health outcomes. Social capital is an important determinant of mental health problems but under-studied in this population. This study investigated the association between social capital and mental health problems among older people living with HIV in China. METHODS: The study was based on the baseline data of a cohort study investigating mental health among older people living with HIV in Sichuan, China during November 2018 to February 2019. Participants were people living with HIV aged ≥50 years living in Sichuan province. Stratified multi-stage cluster sampling was used to recruit participants from 30 communities/towns; 529 out of 556 participants being approached completed the face-to-face interview. Social capital was measured by two validated health-related social capital scales: the Individual and Family scale and the Community and Society scale. Presence of probable depression (CES-D-10 score ≥ 10) and probable anxiety (GAD-7 score ≥ 5) were used as dependent variables. Two-level logistic regression models were applied to examine the association between social capital and probable depression/anxiety. RESULTS: The prevalence of probable depression and probable anxiety was 25.9% (137/529) and 36.3% (192/529), respectively. After adjusting for significant covariates, the individual/family level of social capital was inversely associated with both probable depression (odds ratios (OR): 0.89, 95% CI: 0.84-0.93, p < 0.001) and probable anxiety (OR: 0.90, 95% CI: 0.86-0.95, p < 0.001). The community/society level social capital was associated with probable depression (OR: 0.91, 95% CI: 0.84-0.99, p < 0.001) but not probable anxiety (p > 0.05). CONCLUSIONS: Interventions building up social capital should be considered to improve mental health of older people living with HIV. Some useful strategies include cognitive processing therapy, improving community networking and engagement, and promoting social bonding with neighborhood.

Structural basis for interaction between Mycobacterium smegmatis Ms6564, a TetR family master regulator, and its target DNA.

Master regulators, which broadly affect expression of diverse genes, play critical roles in bacterial growth and environmental adaptation. However, the underlying mechanism by which such regulators interact with their cognate DNA remains to be elucidated. In this study, we solved the crystal structure of a broad regulator Ms6564 in Mycobacterium smegmatis and its protein-operator complex at resolutions of 1.9 and 2.5 Å, respectively. Similar to other typical TetR family regulators, two dimeric Ms6564 molecules were found to bind to opposite sides of target DNA. However, the recognition helix of Ms6564 inserted only slightly into the DNA major groove. Unexpectedly, 11 disordered water molecules bridged the interface of TetR family regulator DNA. Although the DNA was deformed upon Ms6564 binding, it still retained the conformation of B-form DNA. Within the DNA-binding domain of Ms6564, only two amino acids residues directly interacted with the bases of cognate DNA. Lys-47 was found to be essential for the specific DNA binding ability of Ms6564. These data indicate that Ms6564 can bind DNA with strong affinity but makes flexible contacts with DNA. Our study suggests that Ms6564 might slide more easily along the genomic DNA and extensively regulate the expression of diverse genes in M. smegmatis.

Molecular basis of human trace amine-associated receptor 1 activation.

The human trace amine-associated receptor 1 (hTAAR1, hTA1) is a key regulator of monoaminergic neurotransmission and the actions of psychostimulants. Despite preclinical research demonstrating its tractability as a drug target, its molecular mechanisms of activation remain unclear. Moreover, poorly understood pharmacological differences between rodent and human TA1 complicate the translation of findings from preclinical disease models into novel pharmacotherapies. To elucidate hTA1's mechanisms on the molecular scale and investigate the underpinnings of its divergent pharmacology from rodent orthologs, we herein report the structure of the human TA1 receptor in complex with a Gαs heterotrimer. Our structure reveals shared structural elements with other TAARs, as well as with its closest monoaminergic orthologue, the serotonin receptor 5-HT4R. We further find that a single mutation dramatically shifts the selectivity of hTA1 towards that of its rodent orthologues, and report on the effects of substituting residues to those found in serotonin and dopamine receptors. Strikingly, we also discover that the atypical antipsychotic medication and pan-monoaminergic antagonist asenapine potently and efficaciously activates hTA1. Together our studies provide detailed insight into hTA1 structure and function, contrast its molecular pharmacology with that of related receptors, and uncover off-target activities of monoaminergic drugs at hTA1.

Molecular Basis of Human Trace Amine-Associated Receptor 1 Activation.

The human trace amine-associated receptor 1 (hTAAR1, hTA1) is a key regulator of monoaminergic neurotransmission and the actions of psychostimulants. Despite preclinical research demonstrating its tractability as a drug target, its molecular mechanisms of activation remain unclear. Moreover, poorly understood pharmacological differences between rodent and human TA1 complicate the translation of findings from preclinical disease models into novel pharmacotherapies. To elucidate hTA1's mechanisms on the molecular scale and investigate the underpinnings of its divergent pharmacology from rodent orthologs, we herein report the structure of the human TA1 receptor in complex with a Gαs heterotrimer. Our structure reveals shared structural elements with other TAARs, as well as with its closest monoaminergic ortholog, the serotonin receptor 5-HT4R. We further find that a single mutation dramatically shifts the selectivity of hTA1 towards that of its rodent orthologs, and report on the effects of substituting residues to those found in serotonin and dopamine receptors. Strikingly, we also discover that the atypical antipsychotic medication and pan-monoaminergic antagonist asenapine potently and efficaciously activates hTA1. Together our studies provide detailed insight into hTA1 structure and function, contrast its molecular pharmacology with that of related receptors, and uncover off-target activities of monoaminergic drugs at hTA1.

Substrate binding and inhibition of the anion exchanger 1 transporter.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions.

Archaeal DNA replication machinery represents a core version of that found in eukaryotes. However, the proteins essential for the coordination of origin selection and the functioning of DNA polymerase have not yet been characterized in archaea, and they are still being investigated in eukaryotes. In the current study, the Orc1/Cdc6 (SsoCdc6) proteins from the crenarchaeon Sulfolobus solfataricus were found to physically interact with its DNA polymerase B1 (SsoPolB1). These SsoCdc6 proteins stimulated the DNA-binding ability of SsoPolB1 and differentially regulated both its polymerase and nuclease activities. Furthermore, the proteins also mutually regulated their interactions with SsoPolB1. In addition, SsoPolB1c467, a nuclease domain-deleted mutant of SsoPolB1 defective in DNA binding, retains the ability to physically interact with SsoCdc6 proteins. Its DNA polymerase activity could be stimulated by these proteins. We report on a linkage between the initiator protein Orc1/Cdc6 and DNA polymerase in the archaeon. Our present and previous findings indicate that archaeal Orc1/Cdc6 proteins could potentially play critical roles in the coordination of origin selection and cell-cycle control of replication.

Archaeal eukaryote-like serine/threonine protein kinase interacts with and phosphorylates a forkhead-associated-domain-containing protein.

Protein phosphorylation plays an important role in cell signaling. However, in the Archaea, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon Sulfolobus tokodaii strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both in vivo and in vitro. ST1565 preferred Mn(2+) as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45 degrees C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the Archaea, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life.

Characterization of physical and functional interactions between eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase in the hyperthermophilic archaeon Sulfolobus solfataricus.

The roles of Y-family DNA polymerases and the regulation mechanisms are not well defined in Archaea. In this study, we performed in vitro and in vivo characterization of the physical interaction between the archaeon Sulfolobus solfataricus Y-family DNA polymerase (SsoPolY) and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). The effect of SsoCdc6-2 was the strongest, and the three SsoCdc6 proteins were shown to have very different effects on the function of SsoPolY. SsoCdc6-2 inhibited both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it formed a large complex with SsoPolY and stimulated DNA-binding activity on paired primer-template DNA substrates. SsoCdc6-2 and S. solfataricus PCNA (SsoPCNA) showed a cooperative effect on polymerization by SsoPolY on paired DNA templates, but SsoCdc6 reduced the stimulating effect of SsoPCNA on this polymerization on mismatched DNA substrates. Therefore, we uncovered a DNA substrate-dependent SsoCdc6/SsoPolY interaction mechanism. This is the first evidence for a physical and functional linkage between archaeal eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase.

Association between Psychological Factors and Condom Use with Regular and Nonregular Male Sexual Partners among Chinese MSM: A Quantitative Study Based on the Health Belief Model.

OBJECTIVE: The Chinese men who have sex with men (MSM) population is suffering from a high HIV infection rate owing to unprotected anal sex. The Health Belief Model (HBM) has been proven to be an effective frame associated with behavior maintenance. Based on HBM, we analyzed the beliefs associated with consistent condom use behavior with regular and nonregular partners among MSM to better provide targeted interventions and services. METHODS: A study was conducted in Sichuan Province, China, from November 2018 to April 2019, and 801 eligible participants were recruited by snowball sampling. Sociodemographic characteristics, AIDS-related characteristics, sexual behaviors, condom use behavior, and dimensions of HBM were investigated. Univariate, single multivariate, and summary multivariate models were employed to analyze the factors associated with consistent condom use. RESULTS: Of all participants, 39.1% and 53.6% had had anal sex with regular and nonregular partners in the last six months, respectively. Only 56.5% of them had used condoms consistently with regular partners, and only 60% of them had used condoms consistently with nonregular partners. When taking consistent condoms use with regular partners as the dependent variable, the dimensions of perceived threats (ORM = 1.28, 95% CI: 1.10, 1.49), perceived barriers (ORM = 0.70, 95% CI: 0.60, 0.82), self-efficacy (ORM = 1.23, 95% CI: 1.14, 1.32), and cues to action (ORM = 1.21, 95% CI: 1.02, 1.43) showed significant associations with the dependent variable. When taking consistent condoms use with nonregular partners as the dependent variable, the dimensions of perceived barriers (ORM = 0.77, 95% CI: 0.67, 0.89), self-efficacy (ORM = 1.22, 95% CI: 1.13, 1.32), and cues to action (ORM = 1.53, 95% CI: 1.30, 1.80) showed significant associations with the dependent variable. CONCLUSIONS: More attention should be focused on how to decrease the obstructive factors of condom use, how to improve the confidence of condom use, and how to layout more cues to action to promote consistent condom use behavior with regular and nonregular partners during anal sex among Chinese MSM.

Self-efficacy Mediates Perceived Benefits and Barriers of Adherence of Heroin-dependent Patients to Methadone for Addiction Treatment: A Health Belief Model Study.

OBJECTIVE: Although methadone for addiction treatment (MAT) has been widely used in China, the low adherence rate in MAT clinics poses a great challenge. We aimed to investigate the factors related to the adherence of heroin-dependent patients to MAT based on the Health Belief Model (HBM) in Sichuan, China. METHODS: A cross-sectional structured interview was conducted between August and November 2018. Stratified multi-stage sampling was carried out. A total of 581 participants were enrolled from 5 clinics and completed the face-to-face structured interview. Univariate, adjusted logistic regression, multivariate logistic regression analysis and the structural equation modeling (SEM) were employed to explore the association between constructs of HBM and adherence to MAT among heroin-dependent patients. RESULTS: The adherence rate of MAT was 79.3% in the past 6 months. Among all constructs of HBM, self-efficacy (AOR: 1.16, 95% CI: 1.10, 1.22), perceived benefits (AOR: 1.05, 95% CI: 1.00, 1.10) and perceived barriers (AOR: 0.87, 95% CI: 0.77, 0.98) were associated with adherence to MAT. Self-efficacy was directly associated with adherence to MAT (ß = 0.347, P < 0.05). Perceive benefits (ß = 0.276, P < 0.01) and perceived barriers (ß = -0.241, P < 0.05) were directly associated with self-efficacy. However, perceived benefits (ß = 0.096, P < 0.01) and perceived barriers (ß = -0.084, P < 0.01) were only indirectly associated with adherence to MAT. CONCLUSION: The adherence of heroin-dependent patients to MAT can be explained by self-efficacy, perceived benefits and barriers. Self-efficacy plays a significant role as a mediating variable. Future interventions should be considered to improve patients' self-efficacy to MAT.

Relationship between social capital and heroin use behaviors among patients in methadone maintenance treatment in Sichuan Province, China: A cross-sectional study.

Covertly using heroin during methadone maintenance treatment (MMT) is very common among heroin-dependent patients, which has posed threats to the physical health of heroin-dependent patients and social safety. Covertly using heroin may be influenced by many factors, especially social capital. Therefore, we aimed to investigate the relationship between behaviors of covertly using heroin during MMT and social capital heroin-dependent patients in Sichuan Province, China. A cross-sectional study was conducted between October and November 2018, with a total of 581 heroin-dependent patients participating in the study. In addition to socio-demographic characteristics and heroin use related behaviors, the questionnaire also included the measures of social capital: social network (SN), social support (SP), community participation (CP) and social trust (ST). Multivariate logistic regression analyses were used to estimate the association between different measures of social capital and heroin use. The prevalence of covertly using heroin of heroin during MMT was 31.0% among our participants in the 6 months before the study. After adjusting for socio-demographic factors and heroin-use related variables, SN (OR = 0.85, 95% CI: 0.76-0.95), SP (OR = 0.89, 95% CI: 0.83-0.95), and ST (OR = 0.88, 95% CI: 0.81-0.95) were significantly associated with heroin use. Results suggest that social capital may have a protective effect on behavior of covertly using heroin during MMT, which should be consider in the interventions for heroin-dependent patients, in order to reduce the incidence of heroin use during MMT as well as improve the compliance of MMT.