Computational and Structural Biotechnology Journal, 2021
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in so... more Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in some parts of the world. As with other RNA viruses, mutations in the SARS-CoV-2 gene have been continuously evolving. Recently, four variants have been identified, B.1.1.7, B.1.351, P.1 and CAL.20C. These variants appear to be more infectious and transmissible than the original Wuhan-Hu-1 virus. Using a combination of bioinformatics and structural analyses, we show that the new SARS-CoV-2 variants emerged in the background of an already known Spike protein mutation D614G together with another mutation P323L in the RNA polymerase of SARS-CoV-2. The phylogenetic analysis showed that the CAL.20C and B.1.351 shared one common ancestor, whereas the B.1.1.7 and P.1 shared a different ancestor. Structural comparisons did not show any significant difference between the wild-type and mutant ACE2/Spike complexes. Structural analysis indicated that the N501Y mutation may increase hydrophobic interactions at the ACE2/Spike interface. However, reported greater binding affinity of N501Y Spike with ACE2 does not seem to be entirely due to increased hydrophobic interactions, given that Spike mutation R417T in P.1 or K417N in B.1.351 results in the loss of a salt-bridge interaction between ACE2 and S-RBD. The calculated change in free energy did not provide a clear trend of S protein stability of mutations in the variants. As expected, we show that the CAL.20C generally migrated from the west coast to the east coast of the USA. Taken together, the analyses suggest that the evolution of variants and their infectivity is complex and may depend upon many factors.
CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we soug... more CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we sought to understand the requirement for CD40 signaling in the programmed cell death-1 (PD-1) checkpoint and CD28 costimulatory pathways important for maintenance of peripheral tolerance. Blocking either pathway can result in loss of self-tolerance and development of autoimmunity. We found that primary Sjögren’s syndrome (pSS) and autoimmune thyroid diseases (ATDs) that develop spontaneously in CD28-deficient IFN-γ−/− NOD.H-2h4 (CD28−/−) mice required CD40 signaling. Specifically, blockade of CD40L with the anti-CD40L mAb, MR1, inhibited autoantibody production and inflammation in thyroid and salivary gland target tissues. Unexpectedly, however, ATD and pSS in PD-1–deficient IFN-γ−/− NOD.H-2h4 (PD-1−/−) mice developed independently of CD40/CD40L interactions. Treatment with MR1 had no effect and even exacerbated disease development in pSS and ATD, respectively. Most interesting, anti-thyrogl...
Extracellular nucleotides exert various effects in the cardiovascular system. Uridine 5′‐triphosp... more Extracellular nucleotides exert various effects in the cardiovascular system. Uridine 5′‐triphosphate (UTP) regulates G‐protein‐coupled P2Y2 receptor (P2Y2R) activation which causes actin cytoskeletal reorganization. We isolated aortic smooth muscle cells (SMC) from wild‐type (WT) and P2Y2R−/− mice to investigate whether UTP and the P2Y2R modulate expression of low density lipoprotein receptor‐related protein 1 (LRP1) and low density lipoprotein receptor (LDLR). For LRP1 expression, cells were stimulated in the presence or absence of 100 μM UTP overnight. For LDLR mRNA expression, cells were transiently transfected for 24 h with cDNA encoding a hemagglutinin‐tagged WT P2Y2R or a mutant P2Y2R that does not bind filamin‐A (FLN‐A) and treated with 100 μM UTP overnight. Total RNA was isolated, reversed transcribed to cDNA, and mRNA abundance determined by RT‐PCR using the ΔCt method with GAPDH as a control gene. Results show SMC expressing the P2Y2R that lacks the FLN‐A binding domain e...
Extracellular nucleotides exert a large num- ber of physiological effects through activation of P... more Extracellular nucleotides exert a large num- ber of physiological effects through activation of P2Y re- ceptors. We expressed rat P2Y2 (rP2Y2) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining (Ca 2+ )i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y2-GFP receptor internal-
Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unpre... more Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background o...
Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of ... more Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis. In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A1, A2A, A2B and A3 for extracellular adenosine, P2X1-7 receptors that are ATP-gated ion channels and G protein-coupled P2Y1,2,4,6,11,12,13 and 14 receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose. Recent studies have implicated specific P2Y receptor subtypes in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance, where G protein-mediated signaling cascades modulate intracellular ion concentrations and activate downstream protein kinases, Src family kinases as well as numerous mitogen-activated protein kinases. We are honored to contribute to this special issue dedicated to the founder of the field of purinergic signaling, Dr. Geoffrey Burnstock, by reviewing the diverse roles of P2Y receptors in the initiation, progression and metastasis of specific cancers with an emphasis on pharmacological and genetic strategies employed to delineate cell-specific and P2Y receptor subtype-specific responses that have been investigated using in vitro and in vivo cancer models. We further highlight bioinformatic and empirical evidence on P2Y receptor expression in human clinical specimens and cover clinical perspectives where P2Y receptor-targeting interventions may have therapeutic relevance to cancer treatment.
Computational and Structural Biotechnology Journal, 2021
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in so... more Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in some parts of the world. As with other RNA viruses, mutations in the SARS-CoV-2 gene have been continuously evolving. Recently, four variants have been identified, B.1.1.7, B.1.351, P.1 and CAL.20C. These variants appear to be more infectious and transmissible than the original Wuhan-Hu-1 virus. Using a combination of bioinformatics and structural analyses, we show that the new SARS-CoV-2 variants emerged in the background of an already known Spike protein mutation D614G together with another mutation P323L in the RNA polymerase of SARS-CoV-2. The phylogenetic analysis showed that the CAL.20C and B.1.351 shared one common ancestor, whereas the B.1.1.7 and P.1 shared a different ancestor. Structural comparisons did not show any significant difference between the wild-type and mutant ACE2/Spike complexes. Structural analysis indicated that the N501Y mutation may increase hydrophobic interactions at the ACE2/Spike interface. However, reported greater binding affinity of N501Y Spike with ACE2 does not seem to be entirely due to increased hydrophobic interactions, given that Spike mutation R417T in P.1 or K417N in B.1.351 results in the loss of a salt-bridge interaction between ACE2 and S-RBD. The calculated change in free energy did not provide a clear trend of S protein stability of mutations in the variants. As expected, we show that the CAL.20C generally migrated from the west coast to the east coast of the USA. Taken together, the analyses suggest that the evolution of variants and their infectivity is complex and may depend upon many factors.
CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we soug... more CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we sought to understand the requirement for CD40 signaling in the programmed cell death-1 (PD-1) checkpoint and CD28 costimulatory pathways important for maintenance of peripheral tolerance. Blocking either pathway can result in loss of self-tolerance and development of autoimmunity. We found that primary Sjögren’s syndrome (pSS) and autoimmune thyroid diseases (ATDs) that develop spontaneously in CD28-deficient IFN-γ−/− NOD.H-2h4 (CD28−/−) mice required CD40 signaling. Specifically, blockade of CD40L with the anti-CD40L mAb, MR1, inhibited autoantibody production and inflammation in thyroid and salivary gland target tissues. Unexpectedly, however, ATD and pSS in PD-1–deficient IFN-γ−/− NOD.H-2h4 (PD-1−/−) mice developed independently of CD40/CD40L interactions. Treatment with MR1 had no effect and even exacerbated disease development in pSS and ATD, respectively. Most interesting, anti-thyrogl...
Extracellular nucleotides exert various effects in the cardiovascular system. Uridine 5′‐triphosp... more Extracellular nucleotides exert various effects in the cardiovascular system. Uridine 5′‐triphosphate (UTP) regulates G‐protein‐coupled P2Y2 receptor (P2Y2R) activation which causes actin cytoskeletal reorganization. We isolated aortic smooth muscle cells (SMC) from wild‐type (WT) and P2Y2R−/− mice to investigate whether UTP and the P2Y2R modulate expression of low density lipoprotein receptor‐related protein 1 (LRP1) and low density lipoprotein receptor (LDLR). For LRP1 expression, cells were stimulated in the presence or absence of 100 μM UTP overnight. For LDLR mRNA expression, cells were transiently transfected for 24 h with cDNA encoding a hemagglutinin‐tagged WT P2Y2R or a mutant P2Y2R that does not bind filamin‐A (FLN‐A) and treated with 100 μM UTP overnight. Total RNA was isolated, reversed transcribed to cDNA, and mRNA abundance determined by RT‐PCR using the ΔCt method with GAPDH as a control gene. Results show SMC expressing the P2Y2R that lacks the FLN‐A binding domain e...
Extracellular nucleotides exert a large num- ber of physiological effects through activation of P... more Extracellular nucleotides exert a large num- ber of physiological effects through activation of P2Y re- ceptors. We expressed rat P2Y2 (rP2Y2) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining (Ca 2+ )i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y2-GFP receptor internal-
Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unpre... more Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background o...
Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of ... more Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis. In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A1, A2A, A2B and A3 for extracellular adenosine, P2X1-7 receptors that are ATP-gated ion channels and G protein-coupled P2Y1,2,4,6,11,12,13 and 14 receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose. Recent studies have implicated specific P2Y receptor subtypes in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance, where G protein-mediated signaling cascades modulate intracellular ion concentrations and activate downstream protein kinases, Src family kinases as well as numerous mitogen-activated protein kinases. We are honored to contribute to this special issue dedicated to the founder of the field of purinergic signaling, Dr. Geoffrey Burnstock, by reviewing the diverse roles of P2Y receptors in the initiation, progression and metastasis of specific cancers with an emphasis on pharmacological and genetic strategies employed to delineate cell-specific and P2Y receptor subtype-specific responses that have been investigated using in vitro and in vivo cancer models. We further highlight bioinformatic and empirical evidence on P2Y receptor expression in human clinical specimens and cover clinical perspectives where P2Y receptor-targeting interventions may have therapeutic relevance to cancer treatment.
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Papers by Gary Weisman