Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of ...

University of Iowa

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih(-/-)) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk(-/-)) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk(-/-) embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk(s11) variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.

Early myocardial function affects endocardial cushion development in zebrafish.

To determine the reason for the inviability of Saccharomyces cerevisiae with skeletal muscle actin, we introduced into yeast actin the first variant muscle residue from the C-terminal end, H372R. Arg is also found at this position in non-yeast nonmuscle actins. The substitution caused retarded growth on glucose and an inability to use glycerol as a sole carbon source. The mitochondria were clumped and had lost their DNA, the vacuole appeared hypervesiculated, and the actin cytoskeleton became somewhat depolarized. Introduction of the second muscle actin-specific substitution, S365A, rescued these defects. Suppression was also achieved by introducing the four acidic N-terminal residues of muscle actin in place of the two found in yeast actin. The H372R substitution results in an increase in polymerization-dependent fluorescence of Cys-374 pyrene-labeled actin. H372R actin polymerizes slightly faster than wild-type (WT) actin. Yeast actin-related proteins 2 and 3 (Arp2/3) accelerates the polymerization of H372R actin to a much greater extent than WT actin. The two suppressors did not affect the rate of H372R actin polymerization in the absence of an Arp2/3 complex. In contrast, the S365A substitution dampened the rate of Arp2/3 complex-stimulated H372R actin polymerization, and the addition of the four acidic N-terminal residues caused this rate to decrease below that observed with WT actin in the presence of Arp2/3. Structural analysis of the mutations suggests the presence of stringent steric and ionic requirements for the bottom of actin subdomain 1 and also suggests that there is allosteric communication through subdomain 1 within the actin monomer between the N and C termini.

A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.

Pour d&eacute;terminer la raison de la non-viabilit&eacute; de Saccharomyces cerevisiae avec l&#39;actine musculaire squelettique, nous introduit dans l&#39;actine levure le premier r&eacute;sidu de muscle variant de l&#39;extr&eacute;mit&eacute; C-terminale, H372R. ARG se trouve &eacute;galement &agrave; cet endroit en actines non-levure non musculaires. La substitution provoqu&eacute; le retard de croissance relatif au glucose et une incapacit&eacute; &agrave; utiliser le glyc&eacute;rol comme seule source de carbone. Les mitochondries sont group&eacute; en masse compacte et avaient perdu leur ADN, la vacuole est apparu hypervesiculated, et le cytosquelette d&#39;actine est devenu un peu d&eacute;polaris&eacute;. Introduction de la substitution de sp&eacute;cifiques &agrave; l&#39;actine musculaire deuxi&egrave;me, S365A, a sauv&eacute; ces d&eacute;fauts. Suppression a &eacute;t&eacute; &eacute;galement obtenue en introduisant les quatre r&eacute;sidus acides de N-terminal d&#39;actine musculaire en lieu et place les deux trouv&eacute;s en actine de la levure. La substitution de H372R entra&icirc;ne une augmentation fluorescence d&eacute;pendante de polym&eacute;risation de l&#39;actine de pyr&egrave;ne-&eacute;tiquet&eacute; Cys-374. H372R actine se polym&eacute;rise l&eacute;g&egrave;rement plus rapidement que l&#39;actine sauvage (WT). Levure prot&eacute;ines actine 2 et 3 (Arp2/3) acc&eacute;l&egrave;re la polym&eacute;risation de l&#39;actine H372R dans une mesure beaucoup plus grande que l&#39;actine WT. Les deux suppresseurs n&#39;affectent pas le taux de polym&eacute;risation de l&#39;actine H372R en l&#39;absence d&#39;un complexe Arp2/3. En revanche, la substitution de S365A humidifi&eacute; la vitesse de polym&eacute;risation de l&#39;actine H372R de cr&eacute;ation stimul&eacute;e par le complexe Arp2/3, et l&#39;ajout des quatre r&eacute;sidus acides N-terminal a caus&eacute; ce taux diminuer au-dessous de celle observ&eacute;e avec l&#39;actine WT en pr&eacute;sence de Arp2/3. L&#39;analyse structurale des mutations sugg&egrave;re la pr&eacute;sence d&#39;exigences strictes de st&eacute;riques et ioniques pour le bas du sous-domaine de l&#39;actine 1 et sugg&egrave;re aussi qu&#39;il n&#39;y a communication allost&eacute;rique par sous-domaine 1 dans le monom&egrave;re d&#39;actine entre les extr&eacute;mit&eacute;s N et C terminales.

Une substitution de mammif&egrave;res de l&#39;actine en actine (H372R) de la levure provoque un ph&eacute;notype suppressible mitochondries/vacuole.

Per determinare il motivo per il inviability di Saccharomyces cerevisiae con actina muscolo scheletrico, abbiamo introdotto in actina lievito residuo primo variante muscolo dall&#39;estremit&agrave; C-terminale, H372R. Arg inoltre &egrave; trovato in questa posizione in actina non lievito nonmuscle. La sostituzione ha causato la crescita ritardata il glucosio e l&#39;incapacit&agrave; di utilizzare il glicerolo come un&#39;unica fonte di carbonio. I mitocondri sono stati blocchi e aveva perso il loro DNA, il vacuolo &egrave; apparso hypervesiculated, e il citoscheletro di actina &egrave; diventato un po &#39; depolarized. Introduzione della seconda sostituzione specifici dell&#39;actina muscolare, S365A, salvo questi difetti. Soppressione &egrave; stato raggiunto anche grazie all&#39;introduzione di quattro residui acidi N-terminale di actina muscolo al posto di due trovati in actina lievito. La sostituzione di H372R si traduce in un aumento di fluorescenza dipendente dalla polimerizzazione dell&#39;actina con etichetta pirene Cys-374. H372R actina polimerizza leggermente pi&ugrave; veloce di actina di wild-type (WT). 2 Le proteine actina-relative di lievito e 3 (Arp2/3) accelera la polimerizzazione dell&#39;actina H372R in misura molto maggiore rispetto a actina WT. I due soppressori non intaccarono la velocit&agrave; di polimerizzazione dell&#39;actina di H372R in assenza di un complesso Arp2/3. Al contrario, la sostituzione S365A inumidito la velocit&agrave; di polimerizzazione dell&#39;actina di Arp2/3 complessi-stimolato H372R, e l&#39;aggiunta di quattro residui acidi N-terminale ha causato questo tasso a diminuire di sotto che osservate con actina WT in presenza di Arp2/3. Analisi strutturale delle mutazioni suggeriscono la presenza di severi requisiti sterico e ionici, la parte inferiore del sottodominio actina 1 e suggeriscono anche che non c&#39;&egrave; comunicazione allosterica attraverso sottodominio 1 all&#39;interno di monomeri di actina tra i termini N e C.

Una sostituzione di actina dei mammiferi in actina lievito (H372R) provoca un fenotipo sopprimibile mitocondri/vacuolo.

Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two yeast N-terminal negative charges supported viability. The four negative charge muscle N terminus in a muscle subdomain 1 background caused death, but in the same background actin with three N-terminal acidic residues (3Ac/Sub1) led to sick but viable cells. Addition of three muscle subdomain 2 residues (3Ac/Sub12) produced no further deleterious effects. These hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, and mitochondrial and endocytosis defects. 3Ac/Sub1 G-actin exchanged bound epsilonATP more slowly than wild type actin, and the exchange rate for 3Ac/Sub12 was even slower, similar to that for muscle actin. The mutant actins polymerized faster and produced less stable and shorter filaments than yeast actin, the opposite of that expected for muscle actin. Unlike wild type actin, in the absence of unbound ATP, polymerization led to ADP-F-actin, which rapidly depolymerized. Like yeast actin, the hybrid actins activated muscle myosin S1 ATPase activity only about one-eighth as well as muscle actin, despite having essentially a muscle actin-specific myosin-binding site. Finally, the hybrid actins behaved abnormally in a yeast Arp2/3-dependent polymerization assay. Our results demonstrate a unique sensitivity of yeast to actin N-terminal negative charge density. They also provide insight into the role of each domain in the control of the various functions of actin.

Effect of the substitution of muscle actin-specific subdomain 1 and 2 residues in yeast actin on actin function.

A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/K(d), however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (K(d) = 2 microm versus 0.6 microm). These hybrids bound even more weakly to HPF than did yeast actin (K(d) = 5 microm versus 3.2 microm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster k(off) and a 2 times faster k(on). sub12 bound with a 3 times faster k(off) and a 1.5 times slower k(on). Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site.

Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin.

Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction.

Role of intermonomer ionic bridges in the stabilization of the actin filament.

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76-79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.

A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79.

Twenty-two missense mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause thoracic aortic aneurysm and dissection. Limited access to diseased tissue, the presence of multiple unresolvable actin isoforms in the cell, and lack of an animal model have prevented analysis of the biochemical mechanisms underlying this pathology. We have utilized actin from the yeast Saccharomyces cerevisiae, 86% identical to human α-smooth muscle actin, as a model. Two of the known human mutations, N115T and R116Q, were engineered into yeast actin, and their effect on actin function in vivo and in vitro was investigated. Both mutants exhibited reduced ability to grow under a variety of stress conditions, which hampered N115T cells more than R116Q cells. Both strains exhibited abnormal mitochondrial morphology indicative of a faulty actin cytoskeleton. In vitro, the mutant actins exhibited altered thermostability and nucleotide exchange rates, indicating effects of the mutations on monomer conformation, with R116Q the most severely affected. N115T demonstrated a biphasic elongation phase during polymerization, whereas R116Q demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization, and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin despite nearly normal binding affinities of actin for cofilin. The mutant-specific effects on actin behavior suggest that individual mechanisms may contribute to thoracic aortic aneurysm and dissection.

Allele-specific effects of thoracic aortic aneurysm and dissection alpha-smooth muscle actin mutations on actin function.

In the Saccharomyces cerevisiae actin-profilin interface, Ala(167) of the actin barbed end W-loop and His(372) near the C terminus form a clamp around a profilin segment containing residue Arg(81) and Tyr(79). Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function.

Mutant profilin suppresses mutant actin-dependent mitochondrial phenotype in Saccharomyces cerevisiae.

Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physiology and results in the development of distinct congenital disorders. A zebrafish mutant (s434 mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation. We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at the W-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434 mutants show loss of polymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addition, actc1a mutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregulation or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of the W-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTC mutations in human congenital disorders.

The W-loop of alpha-cardiac actin is critical for heart function and endocardial cushion morphogenesis in zebrafish.

More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.

Thoracic aortic aneurysm (TAAD)-causing mutation in actin affects formin regulation of polymerization.

As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catch-slip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds.

Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser-Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals, and we rediagnosed the patient with atypical Baraitser-Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.

Functional analysis of a de novo ACTB mutation in a patient with atypical Baraitser-Winter syndrome.

Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys(113) of one monomer and Glu(195) of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.

Melissa McKane

Department of Biochemistry,
Roy J. and Lucille A. Carver College of Medicine,
Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine

University of Iowa

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Melissa McKane

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry,Roy J. and Lucille A. Carver College of Medicine,Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine

University of Iowa

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Department of Biochemistry,
Roy J. and Lucille A. Carver College of Medicine,
Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine