G-protein Coupled Receptor Protein Research Articles

TIPE proteins control directed migration of human T cells by directing GPCR and lipid second messenger signaling.

Tissue infiltration by circulating leukocytes via directed migration (also referred to as chemotaxis) is a common pathogenic mechanism of inflammatory diseases. G protein-coupled receptors (GPCRs) are essential for sensing chemokine gradients and directing the movement of leukocytes during immune responses. The tumor necrosis factor α-induced protein 8-like (TIPE or TNFAIP8L) family of proteins are newly described pilot proteins that control directed migration of murine leukocytes. However, how leukocytes integrate site-specific directional cues, such as chemokine gradients, and utilize GPCR and TIPE proteins to make directional decisions are not well understood. Using both gene knockdown and biochemical methods, we demonstrated here that 2 human TIPE family members, TNFAIP8 and TIPE2, were essential for directed migration of human CD4+ T cells. T cells deficient in both of these proteins completely lost their directionality. TNFAIP8 interacted with the Gαi subunit of heterotrimeric (α, β, γ) G proteins, whereas TIPE2 bound to PIP2 and PIP3 to spatiotemporally control immune cell migration. Using deletion and site-directed mutagenesis, we established that Gαi interacted with TNFAIP8 through its C-terminal amino acids, and that TIPE2 protein interacted with PIP2 and PIP3 through its positively charged amino acids on the α0 helix and at the grip-like entrance. We also discovered that TIPE protein membrane translocation (i.e. crucial for sensing chemokine gradients) was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm for how human T cells integrate GPCR and phospholipid signaling pathways to control directed migration. These findings have implications for therapeutically targeting TIPE proteins in human inflammatory and autoimmune diseases.

Multiplexed selectivity screening of anti-GPCR antibodies.

G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR subfamilies. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound off-target, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs and for detecting pathological auto-Abs against GPCRs.

A robust antibody discovery platform for difficult-to-express G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods are the most challenging steps in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCR-specific antibodies by obviating difficulties in GPCR preparation. The strategies include (i) conjugation of the P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli, (ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and (iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general-soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting glucagon-like peptide-1 receptor, C-X-C chemokine receptor type 4, and prostaglandin E2 receptor 4 in this study.

Optimization of Discrete Wavelet Transform Feature Representation and Hierarchical Classification of G-Protein Coupled Receptor Using Firefly Algorithm and Particle Swarm Optimization

Ineffective protein feature representation poses problems in protein classification in hierarchical structures. Discrete wavelet transform (DWT) is a feature representation method which generates global and local features based on different wavelet families and decomposition levels. To represent protein sequences, the proper wavelet family and decomposition level must be selected. This paper proposed a hybrid optimization method using particle swarm optimization and the firefly algorithm (FAPSO) to choose the suitable wavelet family and decomposition level of wavelet transformation for protein feature representation. The suggested approach improved on the work of earlier researchers who, in most cases, manually selected the wavelet family and level of decomposition based solely on experience and not on data. The paper also applied the virtual class methods to overcome the error propagation problems in hierarchical classification. The effectiveness of the proposed method was tested on a G-Protein Coupled Receptor (GPCR) protein data set consisting of 5 classes at the family level, 38 classes at the subfamily level, and 87 classes at the sub-subfamily level. Based on the result obtained, the most selected wavelet family and decomposition level chosen to represent GPCR classes by FAPSO are Biorthogonal wavelets and decomposition level 1, respectively. The experimental results show that the representation of GPCR protein using the FAPSO algorithm with virtual classes can yield 97.9%, 86.9%, and 81.3% classification accuracy at the family, subfamily, and sub-subfamily levels, respectively. In conclusion, the result shows that the selection of optimized wavelet family and decomposition level by the FAPSO algorithm, and the virtual class method can be potentially used as the feature representation method and a hierarchical classification method for GPCR protein.

GPCR Protein Feature Representation using Discrete Wavelet Transform and Particle Swarm Optimisation Algorithm

Features play an important role in representing classes in the hierarchy structure, and using unsuitable features will affect classification performance. The discrete wavelet transform (DWT) approach provides the ability to create the appropriate features to represent data. DWT can produce global and local features using different wavelet families and decomposition levels. These two parameters are essential to obtain a suitable representation for classes in the hierarchy structure. This study proposes using a particle swarm optimisation (PSO) algorithm to select the suitable wavelet family and decomposition level for G-protein coupled receptor (GPCR) hierarchical class representation. The results indicate that the PSO algorithm mostly selects Biorthogonal wavelets and decomposition level 2 to represent GPCR protein. Concerning the performance, the proposed method achieved an accuracy of 97.9%, 85.9%, and 77.5% at the family, subfamily, and sub-subfamily levels, respectively.

Probing the structures of G protein-coupled receptors with mass spectrometry-based techniques

G protein-coupled receptors (GPCRs) are key molecules to regulate numerous physiological processes in the human body, and represent one of the largest protein families as targets for drug development for various human diseases. Understanding the structural basis for GPCR activation, signaling and regulatory mechanisms lays the foundation for structure-based drug design. Compared to X-ray crystallography and cryo-electron microscopy which mainly present static snapshots of protein structures, mass spectrometry (MS)-based structural analysis is able to yield complementary information on the conformational ensemble and dynamics of protein machineries and their interactions with ligands or other proteins. Analysis of GPCR structures with multiple MS-based techniques have facilitated the characterization of structural architecture and conformational dynamics of GPCR proteins complexed with specific ligands and/or downstream signaling partners. In this review, we introduce four MS techniques (chemical cross-linking MS, hydrogen/deuterium exchange MS, native MS and fast photochemical oxidation of proteins MS) and illustrate how they are implemented to probe the structures of GPCR proteins and signaling complexes.

Multivariant Transcriptome Analysis Identifies Modules and Hub Genes Associated with Poor Outcomes in Newly Diagnosed Multiple Myeloma Patients.

Simple SummaryMultiple myeloma (MM) is a cancer of plasma cells with a five-year survival rate of 53%. MM is a heterogeneous disease with diverse clinical courses, consistent with the variable efficacy of therapeutic strategies and the development of chemoresistance. We used bioinformatic tools to better understand the molecular mechanisms that underlie failures in the standard treatment of MM with RVD (revlimid, velcade, and dexamethasone). Using an RNA-seq dataset from the MMRF CoMMpass study downloaded from the GDC portal, we identified modules positively correlated to MM vital status. Hub genes from these modules were further grouped based on their biological function and evaluated for association to patient survival.The molecular mechanisms underlying chemoresistance in some newly diagnosed multiple myeloma (MM) patients receiving standard therapies (lenalidomide, bortezomib, and dexamethasone) are poorly understood. Identifying clinically relevant gene networks associated with death due to MM may uncover novel mechanisms, drug targets, and prognostic biomarkers to improve the treatment of the disease. This study used data from the MMRF CoMMpass RNA-seq dataset (N = 270) for weighted gene co-expression network analysis (WGCNA), which identified 21 modules of co-expressed genes. Genes differentially expressed in patients with poor outcomes were assessed using two independent sample t-tests (dead and alive MM patients). The clinical performance of biomarker candidates was evaluated using overall survival via a log-rank Kaplan–Meier and ROC test. Four distinct modules (M10, M13, M15, and M20) were significantly correlated with MM vital status and differentially expressed between the dead (poor outcomes) and the alive MM patients within two years. The biological functions of modules positively correlated with death (M10, M13, and M20) were G-protein coupled receptor protein, cell–cell adhesion, cell cycle regulation genes, and cellular membrane fusion genes. In contrast, a negatively correlated module to MM mortality (M15) was the regulation of B-cell activation and lymphocyte differentiation. MM biomarkers CTAG2, MAGEA6, CCND2, NEK2, and E2F2 were co-expressed in positively correlated modules to MM vital status, which was associated with MM’s lower overall survival.

Post-Translational Modifications of G Protein-Coupled Receptors Revealed by Proteomics and Structural Biology.

G protein–coupled receptors (GPCRs) are a protein superfamily comprising >800 members that regulate numerous cellular and physiologic responses. GPCRs represent the largest class of therapeutic targets with implications in various diseases. Although advances in GPCR structural and pharmacological research have significantly improved our knowledge of GPCR signaling mechanisms, mapping diverse post-translational modifications (PTMs) of GPCR proteins and understanding their regulatory roles have received much less attention. Mass spectrometry-based proteomics has become the most popular technology for profiling protein PTMs in a systematic manner. Herein we provide an overview of PTM types, locations, crosstalk and dynamic regulation for different GPCRs that are characterized using proteomic and/or biochemical approaches. Our main focus is on glycosylation, phosphorylation, ubiquitination and palmitoylation that are known to modulate receptor folding, biosynthesis, trafficking, dimerization and signaling. Furthermore, we discuss the locations of specific PTM sites in the structure of a given GPCR and its signaling complex to highlight the importance of PTM regulation in the molecular basis of GPCRs, which may shed new light on structure-based drug discovery.

Heterologous Expression and Purification of GPCRs.

G protein-coupled receptors (GPCRs) are involved in a variety of human physiological processes and are attractive targets for treating various diseases. Yet, despite the importance as therapeutic targets, only 97 unique GPCR structures have been determined to date. A key challenge in their structural biology study is to obtain adequate protein samples because GPCRs usually have the low expression in native tissues. The in vitro recombinant expression provides the possibility to obtain large quantities of high-quality proteins suitable for three-dimensional structure determination by crystallography or single particle cryo-EM methods. For GPCR protein production, eukaryotic expression systems, such as baculovirus system and mammalian system, are the most widely used. In this chapter, we provide an overview of the methodological approaches on GPCRs expression and purification optimization using insect cells and mammalian cells, which is the prerequisite conditions for structural biology studies.

Pathological AT1R-B2R Protein Aggregation and Preeclampsia.

Preeclampsia is one of the most frequent and severe complications of pregnancy. Symptoms of preeclampsia usually occur after 20 weeks of pregnancy and include hypertension and kidney dysfunction with proteinuria. Up to now, delivery of the infant has been the most effective and life-saving treatment to alleviate symptoms of preeclampsia because a causative treatment does not exist, which could prolong a pregnancy complicated with preeclampsia. Preeclampsia is a complex medical condition, which is attributed to a variety of different risk factors and causes. Risk factors account for insufficient placentation and impaired vasculogenesis and finally culminate in this life-threatening condition of pregnancy. Despite progress, many pathomechanisms and causes of preeclampsia are still incompletely understood. In recent years, it was found that excessive protein complex formation between G-protein-coupled receptors is a common sign of preeclampsia. Specifically, the aberrant heteromerization of two vasoactive G-protein-coupled receptors (GPCRs), the angiotensin II AT1 receptor and the bradykinin B2 receptor, is a causative factor of preeclampsia symptoms. Based on this knowledge, inhibition of abnormal GPCR protein complex formation is an experimental treatment approach of preeclampsia. This review summarizes the impact of pathological GPCR protein aggregation on symptoms of preeclampsia and delineates potential new therapeutic targets.

G protein-coupled receptor-G protein interactions: a single-molecule perspective.

G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants, and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure.

The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

G protein-coupled receptors (GPCRs) are vital for diverse biological functions, including vision, smell, and aging. They are involved in a wide range of diseases, and are among the most important targets of medicinal drugs. Tools that facilitate GPCR studies or GPCR-based technologies or therapies are thus critical to develop. Here we report using our QTY (glutamine, threonine, tyrosine) code to systematically replace 29 membrane-facing leucine, isoleucine, valine, and phenylalanine residues in the transmembrane α-helices of the GPCR CXCR4. This variant, CXCR4QTY29, became more hydrophilic, while retaining the ability to bind its ligand CXCL12. When transfected into HEK293 cells, it inserted into the cell membrane, and initiated cellular signaling. This QTY code has the potential to improve GPCR and membrane protein studies by making it possible to design functional hydrophilic receptors. This tool can be applied to diverse α-helical membrane proteins, and may aid in the development of other applications, including clinical therapies.

Classification of G protein-coupled receptors using attention mechanism

In order to get a better understanding of G Protein-Coupled Receptors (GPCRs) biological function, a precise classification of this kind of receptors in the databases is a real need. To improve the classification accuracy, machine learning algorithms are encountered the major challenge such as the extraction of valuable features. In this paper, we introduce AttentionFam method, which utilizes a novel deep learning architecture to overcome the challenges of prevailing approaches, which are domain specific and computationally intensive. The AttentionFam employs advantages of attention mechanism and representation learning to represent implicitly the features of both the aligned and unaligned GPCR protein sequences. Therefore, feature extraction was carried out from raw protein sequences and thus no sequence alignment methods such as MSA are needed. To evaluate the proposed approach, an extensive set of experiments conducted. The results showed that our proposed method achieved the good accuracy of 97.40%, compared to the state-of-the-art approaches. In addition, it showed better performance in terms of time consumption and less memory for the same data analysis.

A peptide immunoaffinity LC-MS/MS strategy for quantifying the GPCR protein, S1PR1 in human colon biopsies.

Background: S1PR1, a G protein-coupled receptor (GPCR)protein, is a therapeutic target for treatment of autoimmune diseases. As a potential biomarker for drug effect and patient stratification, it is of great significance to measure it in biological samples. However, due to the hydrophobic nature of S1PR1 and the difficulties in extraction and solubilization, as well as low expression levels, quantitative determination of S1PR1 remains challenging. Results: In this work, a peptide immunoaffinity LC-MS/MS method was developed to quantify S1PR1 in biopsy-sized colon samples with an LLOQ of 7.81pM. Conclusion: Peptide immunoaffinity LC-MS/MS based strategy has achieved the desired sensitivity for low abundance S1PR1, and the same strategy could be applied to quantify S1PR1 in multiple species and otherGPCRproteins.

Exploration of potential efficacy markers of Astragali Radix for invigorating Qi based on systematic traditional Chinese medicine

Astragali Radix is the elixir for invigorating Qi, with the effects of invigorating Qi, promoting Yang and nourishing the body. With the deepening researches on the chemical constituents of Astragali Radix, it is used more extensively in clinical application. Based on systematic traditional Chinese medicine theory, in this paper, we characterized the effect of Astragali Radix on invigo-rating Qi from the molecular level, and explored the markers of Astragali Radix on invigorating Qi. Through TCMSP and ChEMBL databases, the active components-targets database of Astragali Radix was constructed to clarify the targets(elements) involved in Astragali Radix's Qi invigorating efficacy system. According to the relationship between the targets, the protein interaction network was constructed, and the network modules(structure) were divided according to the theoretic clustering algorithm molecular complex detection(MCODE), and the boundary of the Qi invigorating efficacy system was defined by the pharmacological function of Astragali Radix. The active components of Astragali Radix for invigorating Qi were characterized from the aspects of composition, target and efficacy. The results showed that eight key components of Astragali Radix, such as hederagenin, quercetin, calycosin, formononetin, jaranol, isorhamnetin, astragalosideⅢ, and 9,10-dimethoxypterocarpan-3-O-β-D-glucoside, could act on eight functional modules composed of 17 key targets, and participate in G-protein coupled receptor protein signaling pathway, regulation of lipid metabolic process, positive regulation of nitrogen compound metabolic process, positive regulation of programmed cell death, fatty acid metabolic process and other biological processes to produce pharmacological effects such as regulating immune function, strengthening heart, protecting myocardial cells, improving material metabolism, and antioxidation effects, thus playing the role of invigorating Qi. Based on the systematic Chinese medicine theory, this study explored the effective markers of Astragali Radix at the level of molecular network, which provided new ideas for the interpretation of the effective substance basis of systematic traditional Chinese medicine and the quality control of traditional Chinese medicine. In the future, it can focus on the compatibility research of these components, and then carry out more in-depth studies on the efficacy of Astragali Radix in invigorating Qi, and strengthen the development of the corresponding pharmacological mechanism and related preparations.

SUN-368 Wnt Inhibition Decreases Trabecular Bone in a Mouse Model of Fibrous Dysplasia

Background: G protein-coupled receptor (GPCR) signaling mediates a wide spectrum of physiological functions, including bone development and remodeling. Fibrous dysplasia (FD) is a common skeletal dysplasia where normal bone and bone marrow are replaced by fibrous tissue and expansile trabecular bone lesions. The craniofacial bones are often involved, leading to pain and facial deformities. FD is a mosaic disease caused by a somatic mutation in the GNAS gene encoding the G-protein alpha subunit (Gsα) that leads to constitutive activation of the Gs signaling pathway. Unfortunately, FD has no effective medical treatments.Major challenges have hampered the development of pharmacologic strategies that specifically target GNAS or the Gsα protein. We previously developed the Col1(2.3)/Rs1 mouse model (Rs1) in which the Gs signaling pathway is activated specifically in bone by an engineered GPCR protein. These mice showed increased trabecular bone formation with loss of marrow space and cortical bone, which strongly resembles human FD (1–4). There was also a dramatic increase in the number of immature osteoblasts present in the FD lesions, suggesting that activation of Gs signaling caused an accumulation of these cells. Our prior studies showed increased Wnt signaling, which may be a major driver of this effect. Furthermore, blocking the Gs signaling could reverse the bone phenotype, providing proof-of-concept for finding drugs that could reverse the phenotype. Therefore, we administered the Wnt inhibitor LGK974, currently used in human clinical trials, to the Rs1 mice to test if the FD lesions could be pharmacologically reversed. Methods: We administered LGK974 in 4-week-old Rs1 and non-Rs1 mice. We used a low dose (5mg/kg) for 8 weeks or high dose (30mg/kg) for 4 weeks. The mice were evaluated by histology and micro computed tomography (micro-CT) for mineral density (mg/cm3), bone volume (mm3), and trabecular thickness (um). Results: LGK974 decreased β-catenin levels in bone on western blots. In the low-dose group, the histology and micro-CT showed no statistically significant differences between drug and control groups. In the high-dose group, the micro-CT showed significantly decreased trabecular bone thickness (p=0.0364, n=3) in the drug-treated group (22±2µm) compared with controls (17±2µm). Furthermore, histology showed resorption of the abnormal bone; however, the fibrocellular infiltrate in the Rs1 mice was still present. Conclusions: Wnt inhibition can lead to decreased fibrous dysplastic bone, but separates abnormal bone formation from the fibrocellular infiltrate. These results provide new insight into understanding interactions between the Wnt and Gs signaling pathways in FD pathogenesis and bone formation.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Physiological functions of a methuselah-like G protein coupled receptor in Lymantria dispar Linnaeus

Insect G protein coupled receptors (GPCRs) have been identified as a highly attractive target for new generation insecticides discovery due to their critical physiological functions. However, few insect GPCRs have been functionally characterized. Here, we cloned the full length of a methuselah-like GPCR gene (Ldmthl1) from the Asian gypsy moth, Lymantria dispar. We then characterized the secondary and tertiary structures of Ldmthl1. We also predicted the global structure of this insect GPCR protein which is composed of three major domains. RNA interference of Ldmthl1 resulted in a reduction of gypsy moths' resistance to deltamethrin and suppressed expression of downstream stress-associated genes, such as P450s, glutathione S transferases, and heat shock proteins. The function of Ldmthl1 was further investigated using transgenic lines of Drosophila melanogaster. Drosophila with overexpression of Ldmthl1 showed significantly longer lifespan than control flies. Taken together, our studies revealed that the physiological functions of Ldmthl1 in L. dispar are associated with longevity and resistance to insecticide stresses. Potentially, Ldmthl1 can be used as a target for new insecticide discovery in order to manage this notorious forest pest.

Gene Expression analysis associated with salt stress in a reciprocally crossed rice population

The rice landrace Horkuch, endemic to the southern saline coast of Bangladesh, is known to have salt tolerance traits and can therefore contribute to a high yielding recipient for breeding purposes. In this study, we reciprocally crossed Horkuch with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage. We looked at tolerant and sensitive F3 families from individual F2 segregating plants and analyzed them for differential gene expressions using RNAseq. In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure. We also observed a strong maternal cytoplasmic effect on gene expression and this was most evident in roots where there was upregulation in functional enrichments related to phosphorylation, electron carriers, transporter and cation transmembrane activities. Stress groups (tolerant and sensitive) response in F3 families were distinctive in both cytoplasmic backgrounds and involved uniquely upregulated genes in tolerant progenies including membrane sensor proteins, enzymes involved with signaling pathways, such as those producing trehalose and G-protein coupled receptor proteins, photosynthesis-related enzymes and golgi body recycling as well as prolamin precursor proteins involved in refolding of proteins. On the other hand, sensitivity was found to be associated with differential upregulation of only a few redox proteins and higher number of apoptosis related genes compared to the tolerant response. Overall, our highly replicated experimental design was powerful and allowed the detection of relatively subtle differential expression. Our future goal is to correlate these expression differences with QTLs in this population, which would help identify the relative importance of specific genetic loci and provide a direct avenue for combining higher levels of salt tolerance with better agronomic traits in rice.

Computer aided protein engineering to enhance the thermo-stability of CXCR1- T4 lysozyme complex

CXCR1, a member in G-protein coupled receptor (GPCR) family, binds to chemokine interleukin-8 (IL-8) specifically and transduces signals to mediate immune and inflammatory responses. Despite the importance of CXCR1, high-resolution structure determination is hindered by the challenges in crystallization. It has been shown that properly designed mutants with enhanced thermostability, together with fusion partner proteins, can be useful to form crystals for GPCR proteins. In this study, in silico protein design was carried out by using homology modeling and molecular dynamics simulations. To validate the computational modeling results, the thermostability of several mutants and the wild type were measured experimentally. Both computational results and experimental data suggest that the mutant L126W has a significant improvement in the thermostability. This study demonstrated that in silico design can guide protein engineering and potentially facilitate protein crystallography research.