Browsing by Subject "Structure"
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Item An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins(Elsevier, 2021) Chen, Qiuyan; Zhuo, Ya; Sharma, Pankaj; Perez, Ivette; Francis, Derek J.; Chakravarthy, Srinivas; Vishnivetskiy, Sergey A.; Berndt, Sandra; Hanson, Susan M.; Zhan, Xuanzhi; Brooks, Evan K.; Altenbach, Christian; Hubbell, Wayne L.; Klug, Candice S.; Iverson, T. M.; Gurevich, Vsevolod V.; Biochemistry and Molecular Biology, School of MedicineG protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP6). IP6 interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP6 on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP6, arrestin-2 forms "infinite" chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP6; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP6, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP6 effect. Because IP6 is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.Item Analyzing Topical Structure in ESL Essays: Not All Topics are Equal(Copyright © Cambridge University Press [BREAK]The original doi for the as-published version of the article is 10.1017/S0272263100009517. To access the doi, open the following DOI site in your browser and cut and paste the doi name where indicated: [LINK]http://dx.doi.org[/LINK][BREAK]Access to the original article may require subscription and authorized logon ID/password. IUPUI faculty/staff/students please check University Library resources before purchasing an article. Questions on finding the original article via our databases? Ask a librarian: [LINK]http://www.ulib.iupui.edu/research/askalibrarian[/LINK]., 1990) Schneider, Melanie; Connor, Ulla, 1948-Topical structure analysis (TSA), a text-based approach to the study of topic in discourse, has been useful in identifying text-based features of coherence. It has also been used to distinguish between essays written by groups of native English speakers with varying degrees of writing proficiency (Witte, 1983a, 1983b). More recently, TSA has distinguished between higher and lower rated ESL essays, but with different results from those found with native speakers of English (Connor & Schneider, 1988). The present study replicated the previous ESL study of two groups of essays written for the TOEFL Test of Written English with three groups of essays. Findings indicate that two topical structure variables, proportions of sequential and parallel topics in the essays, differentiate the highest rated group from the two lower rated groups. We offer explanations for the results and propose that all occurrences of a particular type of topic progression do not contribute equally to the coherence of a text.Item GATING OF THE SENSORY NEURONAL VOLTAGE-GATED SODIUM CHANNEL NAv1.7: ANALYSIS OF THE ROLE OF D3 AND D4 / S4-S5 LINKERS IN TRANSITION TO AN INACTIVATED STATE(2010-04-01T15:56:49Z) Jarecki, Brian W.; Cummins, Theodore R.; Nicol, Grant D.; Oxford, G. S.; Hudmon, Andrew; Schild, John H.Voltage-gated sodium channels (VGSCs) are dynamic membrane-spanning proteins crucial for determining the electrical excitability in nerve and muscle. VGSCs transition, or gate, between opened, closed, and inactivated states, in response to changes in transmembrane potential. Altered VGSC gating can affect electrical communication and is implicated in numerous channelopathies. Nav1.7, a VGSC isoform highly expressed in the peripheral nervous system, plays a unique role in pain perception as evidenced by single point missense mutations causing a spectrum of pain syndromes (inherited erythromelalgia; IEM and paroxysmal extreme pain disorder; PEPD) and nonsense mutations resulting in human insensitivity to pain (CIP). These studies indicate Nav1.7 is critical in pain transduction and, as such, structural perturbations to Nav1.7 affecting conformational stability and response to changes in transmembrane potential have the potential to cause pain. Therefore, the aims of this dissertation were to (1) examine the effects of PEPD mutations on the voltage-dependent properties Nav1.7; (2) investigate the effects Nav1.7 alternative splicing has on the impact of IEM and PEPD mutations; (3) evaluate the effects channelopathies, resulting from slowed inactivation, have on modulating an unusual type of sodium current that flows during membrane repolarization; and (4) determine the structural components involved in stabilizing Nav1.7 inactivation. Standard patch-clamp electrophysiology was used to study changes in channel properties. Results from this dissertation demonstrate that (1) PEPD mutations significantly shift the voltage-dependent properties of Nav1.7 channels, destabilize an inactivated state in a residue specific manner, and render nociceptive neurons hyperexcitable; (2) alternative splicing can functionally impact PEPD; (3) channelopathies, resulting from slowed inactivation in neuronal and muscle VGSC isoforms, increase an unusual sodium conductance that flows during repolarization; and (4) specific residues located in distinct regions of Nav1.7 serve as docking sites to stabilize inactivation at different membrane potentials. Overall, this dissertation answers key questions regarding the molecular mechanics required during inactivation and the biophysical consequences of Nav1.7 mutations implicated in painful disorders. The results of this dissertation are important for a more detailed understanding of pain perception and validate the applicability of studying Nav1.7 for discovery of therapeutic targets for treatment of pain. – Theodore R. Cummins, ChairItem Structural basis for regulated inhibition and substrate selection in yeast glycogen synthase(2017-02) Mahalingan, Krishna Kishore; Hurley, Thomas D.; Elmendorf, Jeffrey; Georgiadis, Millie M.; Roach, Peter J.Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain.Item Structural Basis of Arrestin Binding to Cell Membranes(2024-04) Miller, Kyle Warren; Chen, Qiuyan; Takagi, Yuichiro; Georgiadis, Millie M.; Hurley, Thomas D.Two non-visual arrestins, arrestin2 (Arr2) and arrestin3 (Arr3), selectively interact with activated and phosphorylated G protein-coupled receptors (GPCRs) and play crucial roles in regulating many important physiological processes. Arrestins also engage the lipid bilayer surrounding activated GPCRs, which further potentiates arrestin activation and regulates GPCR trafficking in cells. Because of this, structural and functional understanding of arrestins would provide insight in enhancing arrestin’s GPCR desensitization for various diseases where constitutively active GPCR mutants play a role including congenital endocrine disorders and familial gestational hyperthyroidism. To better understand the membrane binding role of arrestins, we performed in vitro binding assays and demonstrated that Arr2 selectively binds to nanodiscs containing Phosphatidylinositol 4,5-bisphosphate (PIP2) even in the absence of different binding sites. Our cryo-electron microscopy (Cryo-EM) structure of Arr2 in complex with PIP2 nanodisc reveals that multiple structural elements of Arr2, including the finger loop, C domain and C-edge loop, contribute to membrane binding. Eliminating one individual site does not significantly impact Arr2 binding to the nanodisc. Moreover, a preactivated variant of Arr2 shows increased binding to the nanodisc than wildtype. We also labeled four potential membrane binding sites with monobromobimane (mBrB) and detected different levels of fluorescence increase in the presence of nanodisc containing various types of phospholipids. Overall, our study provides detailed structural evidence on how arrestins engage the membrane via multiple contact points and how this can impact arrestin-mediated signaling.Item The Structure of Mood and Anxiety Symptoms in the Perinatal Period(Elsevier, 2023) Miller, Michelle L.; O’Hara, Michael W.; Psychiatry, School of MedicineBackground: The perinatal period is increasingly recognized as a vulnerable time for the development and exacerbation of psychiatric symptoms. Research has often focused on perinatal depression, with much less information on perinatal anxiety. This study examined the psychometric structure of all internalizing (anxiety and mood disorder symptoms) in the perinatal period. Methods: Participants were primarily community adults receiving prenatal care from an academic medical center (N = 246). Participants completed a structured clinical interview using the Interview for Mood and Anxiety Symptoms (IMAS) during pregnancy (28-32 weeks gestation) and the postpartum (6-8 weeks). Clinical interviews dimensionally assessed all current anxiety, mood, and obsessive-compulsive symptoms as well as lifetime psychiatric diagnoses. Results: Confirmatory factor analyses identified three latent factors onto which psychiatric symptoms loaded: Distress (depression, generalized anxiety, irritability, and panic symptoms), Fear (social anxiety, agoraphobia, specific phobia, and obsessive-compulsive symptoms), and Bipolar (mania and obsessive-compulsive symptoms) in both pregnancy and the postpartum. The fit statistics of the models indicated adequate to good fit in both models. Limitations: The IMAS is validated against the DSM-IV-TR rather than the DSM-5 and assessments of psychiatric symptoms were focused only on the current pregnancy. Conclusions: A three-factor model consisting of Distress, Fear and Bipolar latent factors was the best-fitting model in pregnancy and the postpartum period and showed stability across time. The structure of internalizing symptoms has important implications for future perinatal research and can be utilized to guide treatment by highlighting which psychiatric symptoms may be most similar during the perinatal period.Item Transthyretin: a review from a structural perspective(Springer, 2001) Hamilton, J. A.; Benson, M. D.; Biochemistry and Molecular Biology, School of MedicineTransthyretin (formerly called prealbumin) plays important physiological roles as a transporter of thyroxine and retinol-binding protein. X-ray structural studies have provided information on the active conformation of the protein and the site of binding of both ligands. Transthyretin is also one of the precursor proteins commonly found in amyloid deposits. Both wild-type and single-amino-acid-substituted variants have been identified in amyloid deposits, the variants being more amyloidogenic. Sequencing of the gene and the resulting production of a transgenic mouse model have resulted in progress toward solving the mechanism of amyloid formation and detecting the variant gene in individuals at risk.