Indiana University-Purdue University Indianapolis (IUPUI) / Insulin resistance, a hallmark of type 2 diabetes (T2D), is associated with accompanying derangements such as hyperinsulinemia that promote the progression of insulin resistance, yet a mechanism(s) is imperfectly understood. Data have demonstrated that hyperinsulinemia promotes insulin resistance as evidenced by diminished ability of insulin to mobilize glucose transporter GLUT4 to the plasma membrane (PM). We found that loss of PM phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure contributes to hyperinsulinemia-induced insulin resistance. We tested if increased glucose flux through hexosamine biosynthesis pathway (HBP) causes dysregulation of PM components necessary for GLUT4 translocation. Increased HBP activity was detected in 3T3-L1 adipocytes cultured in hyperinsulinemia (5 nM Ins; 12 h) and also 2 mM glucosamine (GlcN), a distal HBP activator, inducing losses of PM PIP2 and F-actin. In accordance with HBP flux directly weakening PIP2/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored F-actin and insulin responsiveness. Furthermore, less invasive challenges with glucose led to PIP2/F-actin dysregulation. New findings support a negative correlation between PM cholesterol accrual, PIP2/F-actin structure and GLUT4 regulation. These data stemmed from parallel study aimed at understanding the antidiabetic mechanism of the nutrient chromium (Cr3+). We found that chromium picolinate (CrPic) enhanced insulin-stimulated GLUT4 trafficking via reduction in PM cholesterol. In line with glucose/cholesterol toxicity findings, we demonstrated that therapeutic effects of CrPic occurred solely in adipocytes with increased HBP activity and a concomitant elevation in PM cholesterol. Mechanistically, data are consistent with a role of AMP-activated protein kinase (AMPK) in CrPic action. These data show that CrPic increases AMPK activity and perhaps suppresses cholesterol synthesis via distal phosphorylation and inactivation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), a rate-limiting enzyme in cholesterol synthesis. Continued study of the consequence of increased HBP activity revealed alterations in cholesterogenic transcription factors – Sp1, SREBP-1, and NFY – with Sp1 showing a significant increase in O-linked glycosylation. Consistent with Sp1 modification eliciting maximal transcriptional activation of SREBP-1, Hmgr mRNA was significantly enhanced. In conclusion, these data are consistent with a central role of PM cholesterol in glucose transport and suggest perturbations in this lipid have a contributory role in developing insulin resistance.

Indiana University-Purdue University Indianapolis (IUPUI) / Our ability to hear relies on sensory cells found in the inner ear that transduce sound into biological signals. Microvilli-like protrusions called stereocilia are bundled on the apical surfaces of these cells and allow them to respond to sound-evoked vibrations. The architecture of the stereocilia bundle is highly patterned to ensure normal hearing. Filaments of polymerized actin proteins are bundled in parallel into large cylindrical structures that define the dimensions of stereocilia. This network is then anchored to the cell by inserting into another actin-based structure called the cuticular plate, which forms a gel-like structure and facilitates the mechanical properties of the bundle. The shape of the bundle is determined through tissue-level and intrinsic polarization signaling pathways. Auditory brainstem-evoked response testing, immunofluorescence imaging, scanning electron microscopy, and biochemical labeling techniques were used to study how the ADF/cofilin family of actin filament severing and depolymerizing proteins contributes to the development of the stereocilia bundle. Loss of these proteins disrupts the normal bundle patterning process, changes the lengths and widths of stereocilia, and alters the regulation of filament ends near the ion channel at stereocilia tips that is responsible for mechanotransduction. The activity of this channel regulates ADF/cofilins and the actin at stereocilia tips. Aberrant actin growth in actin networks beneath the stereocilia bundle influences the bundle patterning process, causes dysmorphic bundles to form. This work identifies that ADF/cofilins are necessary during auditory sensory cell development to facilitate normal bundle patterning and establishes this protein family as a molecular link between mechanotransduction and stereocilia bundle maturation.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第12780号 / 論生博第6号 / 新制||生||38(附属図書館) / 30763 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 豊島 文子, 教授 松本 智裕 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21401号 / 生博第402号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 見学 美根子, 教授 上村 匡, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23334号 / 生博第452号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 渡邊 直樹, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Diseases of heart muscle, such as hypertrophic and dilated cardiomyopathy, are often caused by mutations in proteins of the sarcomere, including actin, troponin, tropomyosin, and myosin. The molecular mechanisms of disease-causing mutations remain unclear because the process of cardiac muscle contraction and the corresponding mutational insults are incompletely defined. To elucidate the underlying mechanisms of cardiac muscle contraction, its regulation, and the effects of disease-causing mutations, the structures of sarcomeric protein assemblies must first be solved. In this dissertation we use interdisciplinary structural biology techniques, including cryo-electron microscopy (cryo-EM), protein-protein docking, and molecular dynamics simulations to investigate the interactions between actin, tropomyosin, and myosin. This structural work is foundational in identifying the molecular effects of mutations. In the first project, we present a novel cryo-EM structure of the cardiac-isoform actomyosin-tropomyosin complex. This structure, which utilizes bovine masseter β-myosin, provides the foundation for understanding the molecular effects of cardiomyopathy-causing mutations that occur at the actomyosin interface. Furthermore, by pairing our structure with protein-protein docking methods and molecular dynamics simulations, we identify complementary and periodic electrostatic interactions between the myosin surface loop 4 and tropomyosin. We hypothesize that these interactions are essential in switching between contraction- and relaxation-mediating states. In a follow up study, we test our myosin loop 4 hypotheses by creating a human cardiac β-myosin all-glycine loop 4 mutant, which abolishes nearly all electrostatic interactions between myosin and tropomyosin. After designing the mutant, we solve the cryo-EM structures of the wild-type and mutant actin-myosin-tropomyosin complexes to high resolution. Our structures confirm that the loop 4 mutant abolishes its interaction with tropomyosin and suggests that the tropomyosin cable on mutant actomyosin filaments is shifted to a new position. Subsequent molecular dynamics simulations corroborate our cryo-EM finding that tropomyosin on mutant actomyosin is displaced from the wild type position. Interaction energy calculations derived from these simulations suggest that the mutant position is significantly less stable than the wild-type. This work provides further evidence that loop 4 interactions are key in stabilizing tropomyosin position during contraction. Finally, to extend our work on the human cardiac actomyosin-tropomyosin complex, we provide insights into the ADP release step of the cardiac β-myosin kinetic cycle. Here, we use a composite method of helical and single-particle cryo-EM reconstruction techniques to solve the structures of the human cardiac actin-myosin-tropomyosin filament in the presence and absence of ADP-Mg2+. This work elucidates the structural basis of cardiac β-myosin ADP release and provides insight into the force-sensing mechanism of the cardiac motor. Lastly, we use our structures to probe how cardiomyopathy-causing mutations potentially disrupt the ADP-to-rigor transition, leading to altered myosin contractility. Overall, the structures solved in this dissertation generate fundamental understanding about the function of the cardiac thin filament and the motor protein, myosin. Moreover, this research provides a framework that connects the initial molecular insults of mutations to the disruption of proper regulation that leads to pathological progression. / 2023-08-24T00:00:00Z

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