ABSTRACT
In this report, we describe incremental changes, during a 2‐year period at a single center with the administration of maintenance infliximab infusions. Given practice‐driven changes ...consisting of 1‐hour infusions and omission of premedications, we aimed to investigate if these changes contributed to severe infusion reactions. We reviewed approximately 900 infliximab infusions in a pediatric ambulatory infusion center from January 1, 2014, to December 31, 2015, for severe adverse reactions requiring either rescue epinephrine or a code blue or “rapid response” activation. In 2015, these practice changes resulted in a 51% decrease in total infusion hours (1281 to 630 infusion hours), despite a 9% increase in total number of infusions. No increase in severe adverse events associated with either rapid 1‐hour infusion or omission of premedications. Our findings highlight a quality‐improvement opportunity to standardize infliximab infusions to streamline care in an ambulatory setting.
Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins ...hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Gα when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Gα, RGS domain binding consequently accelerates Gα-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Gα substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Gα selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Gα complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Gα substrate, suggests that unique structural determinants specific to particular RGS protein/Gα pairings exist and could be used to achieve selective inhibition by small molecules.
ABSTRACTIn this report, we describe incremental changes, over a two year period at a single center with the administration of maintenance infliximab infusions. Given practice-driven changes ...consisting of one-hour infusions and omission of pre-medications, we aimed to investigate if these changes contributed to severe infusion reactions. We reviewed approximately 900 infliximab infusions in a pediatric ambulatory infusion center from January 1, 2014-December 31, 2015 for severe adverse reactions requiring either rescue epinephrine or a code blue or ‘rapid response’ activation. In 2015, these practice changes resulted in a 51% decrease in total infusion hours (1281 to 630 infusion hours), despite a 9% increase in total number of infusions. No increase in severe adverse events associated with either rapid 1-hour infusion or omission of pre-medications. Our findings highlight a quality-improvement opportunity to standardize infliximab infusions to streamline care in an ambulatory setting.
“Regulator of G-protein signaling” (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Gα ...subunits (known as GTPase-accelerating protein or “GAP” activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Gαq subunits. As many vasoconstrictive hormones signal via Gq heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Gαq over Gαi/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Gαi-directed binding and GAP activities without perturbing its association with Gαq. Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Gαi at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Gα complexes highlighted the roles of these residues in wild type RGS2 that weaken Gαi subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Gαq GAP that modulates cardiovascular function.
Surface plasmon resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected ...across the sensor surface. This chapter focuses on high-affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction.
Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human ...Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.
The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the ³⁵Sguanosine 5'-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay ...requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg(2+) concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.
RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for ...specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Gα and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on Gαi subunits and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Gαi1(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Gαi1(R178M/A326S), the authors developed a Transcreener® fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling.
âRegulator of G-protein signalingâ (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling
via their ability to increase the intrinsic GTP hydrolysis rate of Gα ...subunits (known as GTPase-accelerating protein or âGAPâ
activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Gα q subunits. As many vasoconstrictive hormones signal via G q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension.
However, to date the particular structural features within RGS2 determining its selectivity for Gα q over Gα i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Gα i -directed binding and GAP activities without perturbing its association with Gα q . Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic
form of Gα i at 2.8-à resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Gα complexes highlighted
the roles of these residues in wild type RGS2 that weaken Gα i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern
cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a
potent and selective Gα q GAP that modulates cardiovascular function.
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