Constitutive activation of T cells by γ2-herpesviral GPCR through the interaction with cellular CXCR4.

Members of the herpesviral family use multiple strategies to hijack infected host cells and exploit cellular signaling for their pathogenesis and latent infection. Among the most intriguing weapons in the arsenal of pathogenic herpesviruses are the constitutively active virally-encoded G protein-coupled receptors (vGPCRs). Even though vGPCRs contribute to viral pathogenesis such as immune evasion and proliferative disorders, the molecular details of how vGPCRs continuously activate cellular signaling are largely unknown. Here, we report that the vGPCR of Herpesvirus saimiri (HVS), an oncogenic γ2-herpesvirus, constitutively activates T cells via a heteromeric interaction with cellular CXCR4. Constitutive T cell activation also occurs with expression of the vGPCR of Kaposi's sarcoma-associated herpesvirus (KSHV), but not the vGPCR of Epstein-Barr virus. Expression of HVS vGPCR down-regulated the surface expression of CXCR4 but did not induce the degradation of the chemokine receptor, suggesting that vGPCR/CXCR4 signaling continues in cytosolic compartments. The physical association of vGPCR with CXCR4 was demonstrated by proximity ligation assay as well as immunoprecipitation. Interestingly, the constitutive activation of T cells by HVS vGPCR is independent of proximal T cell receptor (TCR) signaling molecules, such as TCRß, Lck, and ZAP70, whereas CXCR4 silencing by shRNA abolished T cell activation by vGPCRs of HVS and KSHV. Furthermore, previously identified inactive vGPCR mutants failed to interact with CXCR4. These findings on the positive cooperativity of vGPCR with cellular CXCR4 in T cell activation extend our current understanding of the molecular mechanisms of vGPCR function and highlight the importance of heteromerization for GPCR activity.

When loss-of-function is loss of function: assessing mutational signatures and impact of loss-of-function genetic variants.

MOTIVATION: Loss-of-function genetic variants are frequently associated with severe clinical phenotypes, yet many are present in the genomes of healthy individuals. The available methods to assess the impact of these variants rely primarily upon evolutionary conservation with little to no consideration of the structural and functional implications for the protein. They further do not provide information to the user regarding specific molecular alterations potentially causative of disease. RESULTS: To address this, we investigate protein features underlying loss-of-function genetic variation and develop a machine learning method, MutPred-LOF, for the discrimination of pathogenic and tolerated variants that can also generate hypotheses on specific molecular events disrupted by the variant. We investigate a large set of human variants derived from the Human Gene Mutation Database, ClinVar and the Exome Aggregation Consortium. Our prediction method shows an area under the Receiver Operating Characteristic curve of 0.85 for all loss-of-function variants and 0.75 for proteins in which both pathogenic and neutral variants have been observed. We applied MutPred-LOF to a set of 1142 de novo vari3ants from neurodevelopmental disorders and find enrichment of pathogenic variants in affected individuals. Overall, our results highlight the potential of computational tools to elucidate causal mechanisms underlying loss of protein function in loss-of-function variants. AVAILABILITY AND IMPLEMENTATION: http://mutpred.mutdb.org. CONTACT: predrag@indiana.edu.

Feedback regulation via AMPK and HIF-1 mediates ROS-dependent longevity in Caenorhabditis elegans.

Mild inhibition of mitochondrial respiration extends the lifespan of many species. In Caenorhabditis elegans, reactive oxygen species (ROS) promote longevity by activating hypoxia-inducible factor 1 (HIF-1) in response to reduced mitochondrial respiration. However, the physiological role and mechanism of ROS-induced longevity are poorly understood. Here, we show that a modest increase in ROS increases the immunity and lifespan of C. elegans through feedback regulation by HIF-1 and AMP-activated protein kinase (AMPK). We found that activation of AMPK as well as HIF-1 mediates the longevity response to ROS. We further showed that AMPK reduces internal levels of ROS, whereas HIF-1 amplifies the levels of internal ROS under conditions that increase ROS. Moreover, mitochondrial ROS increase resistance to various pathogenic bacteria, suggesting a possible association between immunity and long lifespan. Thus, AMPK and HIF-1 may control immunity and longevity tightly by acting as feedback regulators of ROS.

Rampant exchange of the structure and function of extramembrane domains between membrane and water soluble proteins.

Of the membrane proteins of known structure, we found that a remarkable 67% of the water soluble domains are structurally similar to water soluble proteins of known structure. Moreover, 41% of known water soluble protein structures share a domain with an already known membrane protein structure. We also found that functional residues are frequently conserved between extramembrane domains of membrane and soluble proteins that share structural similarity. These results suggest membrane and soluble proteins readily exchange domains and their attendant functionalities. The exchanges between membrane and soluble proteins are particularly frequent in eukaryotes, indicating that this is an important mechanism for increasing functional complexity. The high level of structural overlap between the two classes of proteins provides an opportunity to employ the extensive information on soluble proteins to illuminate membrane protein structure and function, for which much less is known. To this end, we employed structure guided sequence alignment to elucidate the functions of membrane proteins in the human genome. Our results bridge the gap of fold space between membrane and water soluble proteins and provide a resource for the prediction of membrane protein function. A database of predicted structural and functional relationships for proteins in the human genome is provided at sbi.postech.ac.kr/emdmp.

Preoperative Factors Affecting Graft Survival After ABO-incompatible Adult Liver Transplantation.

BACKGROUND: Although ABO-incompatible liver transplantation (ABOi LT) has undergone remarkable progress, the prognostic factors are poorly understood. This study aimed to elucidate the preoperative factors affecting graft survival after ABOi LT. METHODS: Patients who underwent ABOi LT between January 2012 and December 2020 at a single institution in South Korea were retrospectively reviewed. A total of 146 recipients, including 34 patients with graft loss, were analyzed. RESULTS: In the multivariate Cox proportional hazard model, recipient age (≥55 y; hazard ratio, 2.47; 95% confidence interval, 1.18-5.19; P = 0.017) and donor ABO type (donor A, hazard ratio, 3.12; 95% confidence interval, 1.33-7.33; P = 0.009) were significantly associated with an increased risk of graft loss. The most common cause of graft loss was recipient death due to bacterial infection (15/34, 44.1%). Both recipient age and donor ABO type were associated with an increased risk of recipient death due to bacterial infections. The incidence of complications after ABOi LT, including antibody-mediated rejection and diffuse intrahepatic biliary stricture, did not differ according to recipient age or donor ABO type. CONCLUSIONS: These findings suggest that recipient age and donor ABO type should be considered when preparing for ABOi LT. Careful monitoring and care after transplantation are required for recipients with preoperative risk factors.

Poor Mobilization-Associated Factors in Autologous Hematopoietic Stem Cell Harvest.

Peripheral blood stem cell transplantation (PBSCT) is an important therapeutic measure for both hematologic and non-hematologic diseases. For PBSCT to be successful, sufficient CD34+ cells need to be mobilized and harvested. Although risk factors associated with poor mobilization in patients with hematologic diseases have been reported, studies of patients with non-hematologic diseases and those receiving plerixafor are rare. To identify factors associated with poor mobilization, data from autologous PBSC harvest (PBSCH) in 491 patients were retrospectively collected and analyzed. A multivariate analysis revealed that in patients with a hematologic disease, an age older than 60 years (odds ratio [OR] 1.655, 95% confidence interval [CI] 1.049-2.611, p = 0.008), the use of myelotoxic agents (OR 4.384, 95% CI 2.681-7.168, p < 0.001), and a low platelet count (OR 2.106, 95% CI 1.205-3.682, p = 0.009) were associated with poor mobilization. In patients with non-hematologic diseases, a history of radiation on the pelvis/spine was the sole associated factor (OR 12.200, 95% CI 1.934-76.956, p = 0.008). Among the group of patients who received plerixafor, poor mobilization was observed in 19 patients (19/134, 14.2%) and a difference in the mobilization regimen was noted among the good mobilization group. These results show that the risk factors for poor mobilization in patients with non-hematologic diseases and those receiving plerixafor differ from those in patients with hematologic diseases; as such, non-hematologic patients require special consideration to enable successful PBSCH.

Protein design by fusion: implications for protein structure prediction and evolution.

Domain fusion is a useful tool in protein design. Here, the structure of a fusion of the heterodimeric flagella-assembly proteins FliS and FliC is reported. Although the ability of the fusion protein to maintain the structure of the heterodimer may be apparent, threading-based structural predictions do not properly fuse the heterodimer. Additional examples of naturally occurring heterodimers that are homologous to full-length proteins were identified. These examples highlight that the designed protein was engineered by the same tools as used in the natural evolution of proteins and that heterodimeric structures contain a wealth of information, currently unused, that can improve structural predictions.

Molecular evolution of protein conformational changes revealed by a network of evolutionarily coupled residues.

An improved understanding of protein conformational changes has broad implications for elucidating the mechanisms of various biological processes and for the design of protein engineering experiments. Understanding rearrangements of residue interactions is a key component in the challenge of describing structural transitions. Evolutionary properties of protein sequences and structures are extensively studied; however, evolution of protein motions, especially with respect to interaction rearrangements, has yet to be explored. Here, we investigated the relationship between sequence evolution and protein conformational changes and discovered that structural transitions are encoded in amino acid sequences as coevolving residue pairs. Furthermore, we found that highly coevolving residues are clustered in the flexible regions of proteins and facilitate structural transitions by forming and disrupting their interactions cooperatively. Our results provide insight into the evolution of protein conformational changes and help to identify residues important for structural transitions.

MON-2, a Golgi protein, mediates autophagy-dependent longevity in Caenorhabditis elegans.

The Golgi apparatus plays a central role in trafficking cargoes such as proteins and lipids. Defects in the Golgi apparatus lead to various diseases, but its role in organismal longevity is largely unknown. Using a quantitative proteomic approach, we found that a Golgi protein, MON-2, was up-regulated in long-lived Caenorhabditis elegans mutants with mitochondrial respiration defects and was required for their longevity. Similarly, we showed that DOP1/PAD-1, which acts with MON-2 to traffic macromolecules between the Golgi and endosome, contributed to the longevity of respiration mutants. Furthermore, we demonstrated that MON-2 was required for up-regulation of autophagy, a longevity-associated recycling process, by activating the Atg8 ortholog GABARAP/LGG-1 in C. elegans. Consistently, we showed that mammalian MON2 activated GABARAPL2 through physical interaction, which increased autophagic flux in mammalian cells. Thus, the evolutionarily conserved role of MON2 in trafficking between the Golgi and endosome is an integral part of autophagy-mediated longevity.

Inferring the molecular and phenotypic impact of amino acid variants with MutPred2.

Identifying pathogenic variants and underlying functional alterations is challenging. To this end, we introduce MutPred2, a tool that improves the prioritization of pathogenic amino acid substitutions over existing methods, generates molecular mechanisms potentially causative of disease, and returns interpretable pathogenicity score distributions on individual genomes. Whilst its prioritization performance is state-of-the-art, a distinguishing feature of MutPred2 is the probabilistic modeling of variant impact on specific aspects of protein structure and function that can serve to guide experimental studies of phenotype-altering variants. We demonstrate the utility of MutPred2 in the identification of the structural and functional mutational signatures relevant to Mendelian disorders and the prioritization of de novo mutations associated with complex neurodevelopmental disorders. We then experimentally validate the functional impact of several variants identified in patients with such disorders. We argue that mechanism-driven studies of human inherited disease have the potential to significantly accelerate the discovery of clinically actionable variants.

KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain.

PDZ domain-containing proteins (PDZ proteins) act as scaffolds for protein-protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN-4, a PDZ domain-containing microtubule-associated serine-threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin-4 is required for the long lifespan of daf-2/insulin/IGF-1 receptor mutants. We then show that neurons are crucial tissues for the longevity-promoting role of kin-4. We find that the PDZ domain of KIN-4 binds PTEN, a key factor for the longevity of daf-2 mutants. Moreover, the interaction between KIN-4 and PTEN is essential for the extended lifespan of daf-2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age-related diseases in mammals through their interaction with PTEN.

The effect of multimorbidity on the gap between global and age-comparative self-rated health scores among the Korean elderly.

Most of the behavior change models regard perceived health status as a motivation for healthy behaviors or chronic disease self-management. The aim of this study was to examine the association between the number of chronic diseases and the difference between global and age-comparative self-rated health scores (GSRH and ASRH). We used national representative survey data pertaining to the elderly in 2011 from the Korea Institute for Health and Social Affairs. In total, 10,003 participants (≥60 years old) were selected from those who had completed the survey in 2008. Multinomial logistic regression was used to estimate relative risk ratios (RRR) with 95% confidence intervals. Demographic factors, socioeconomic status, social connection, and healthy life style were adjusted. Individuals with many chronic diseases were more likely to have a positive gap, resulting in a better ASRH score relative to GSRH (p for trend <0.001): 1-2 diseases (RRR = 1.30, 95% CI = 1.07-1.57), 3-4 diseases (RRR = 1.90, 95% CI = 1.55-2.32), and ≥5 diseases (RRR = 1.75, 95% CI = 1.39-2.20). In addition, the association between the number of chronic diseases and a positive gap varied by sex and living area. Our results suggest that a positive gap between GSRH and ASRH that indicates an overestimated age-comparative health, was associated with the number of chronic diseases. Female or urban-living people had stronger associations. Further research is needed to understand how the gap between GSRH and ASRH could be an alternative measure of SRH and a predictor of major health outcomes.

Comprehensive Analyses of Tissue-Specific Networks with Implications to Psychiatric Diseases.

Recent advances in genome sequencing and "omics" technologies are opening new opportunities for improving diagnosis and treatment of human diseases. The precision medicine initiative in particular aims at developing individualized treatment options that take into account individual variability in genes and environment of each person. Systems biology approaches that group genes, transcripts and proteins into functionally meaningful networks will play crucial role in the future of personalized medicine. They will allow comparison of healthy and disease-affected tissues and organs from the same individual, as well as between healthy and disease-afflicted individuals. However, the field faces a multitude of challenges ranging from data integration to statistical and combinatorial issues in data analyses. This chapter describes computational approaches developed by us and the others to tackle challenges in tissue-specific network analyses, with the main focus on psychiatric diseases.

Metazoans evolved by taking domains from soluble proteins to expand intercellular communication network.

A central question in animal evolution is how multicellular animals evolved from unicellular ancestors. We hypothesize that membrane proteins must be key players in the development of multicellularity because they are well positioned to form the cell-cell contacts and to provide the intercellular communication required for the creation of complex organisms. Here we find that a major mechanism for the necessary increase in membrane protein complexity in the transition from non-metazoan to metazoan life was the new incorporation of domains from soluble proteins. The membrane proteins that have incorporated soluble domains in metazoans are enriched in many of the functions unique to multicellular organisms such as cell-cell adhesion, signaling, immune defense and developmental processes. They also show enhanced protein-protein interaction (PPI) network complexity and centrality, suggesting an important role in the cellular diversification found in complex organisms. Our results expose an evolutionary mechanism that contributed to the development of higher life forms.

OASIS: online application for the survival analysis of lifespan assays performed in aging research.

BACKGROUND: Aging is a fundamental biological process. Characterization of genetic and environmental factors that influence lifespan is a crucial step toward understanding the mechanisms of aging at the organism level. To capture the different effects of genetic and environmental factors on lifespan, appropriate statistical analyses are needed. METHODOLOGY/PRINCIPAL FINDINGS: We developed an online application for survival analysis (OASIS) that helps conduct various novel statistical tasks involved in analyzing survival data in a user-friendly manner. OASIS provides standard survival analysis results including Kaplan-Meier estimates and mean/median survival time by taking censored survival data. OASIS also provides various statistical tests including comparison of mean survival time, overall survival curve, and survival rate at specific time point. To visualize survival data, OASIS generates survival and log cumulative hazard plots that enable researchers to easily interpret their experimental results. Furthermore, we provide statistical methods that can analyze variances among survival datasets. In addition, users can analyze proportional effects of risk factors on survival. CONCLUSIONS/SIGNIFICANCE: OASIS provides a platform that is essential to facilitate efficient statistical analyses of survival data in the field of aging research. Web application and a detailed description of algorithms are accessible from http://sbi.postech.ac.kr/oasis.

Bioinformatic approaches for the structure and function of membrane proteins.

Membrane proteins play important roles in the biology of the cell, including intercellular communication and molecular transport. Their well-established importance notwithstanding, the high-resolution structures of membrane proteins remain elusive due to difficulties in protein expression, purification and crystallization. Thus, accurate prediction of membrane protein topology can increase the understanding of membrane protein function. Here, we provide a brief review of the diverse computational methods for predicting membrane protein structure and function, including recent progress and essential bioinformatics tools. Our hope is that this review will be instructive to users studying membrane protein biology in their choice of appropriate bioinformatics methods.