• Alternative splicing dysregulation in mental disorders

      Glatt, Stephen; Cohen, Ori S (2014)
      The brain's ability to adapt ultimately depends on the efficiency with which neuronal connections are made, destroyed, or manipulated. This connectivity is largely controlled by synaptic plasticity, which creates, strengthens, or weakens signals that are necessary for appropriate functioning of the organism. This constant rewiring allows an organism to learn, mature, and cope with the ever-changing environment. However, this rewiring is dependent on the ability to make new proteins, which highlights the importance of transcription, translation, and post-translational modification in the process of synaptic plasticity. Among these cellular functions, transcription plays a key role in providing the necessary variability that is required to regulate neurodevelopment and cognitive behaviors. During transcription, alternative splicing regulates the contents of transcriptomic elements by cutting and stitching the transcribed pre-mRNA and adjusting the configuration of the mature mRNA(s) to meet the necessary cellular requirements. Therefore, it is conceivable that alternative splicing abnormalities can result in inappropriate adjustment of the transcriptome and result in pathological adaptation. In this dissertation, I review the evidence of dysfunctional gene splicing in neuropsychiatric disorders. Then I evaluate the extent of alternative splicing in an animal model for social interaction. This model utilizes valproic acid exposure at a critical developmental period to illicit significant and long-lasting changes in social interaction behavior. Next, I explore the abundance and types of alternative-splicing dysregulationin postmortembrain tissue samples from schizophrenia patients as compared to non-psychotic comparison subjects. Finally, I describe the mechanisms by which a schizophrenia-associated polymorphism in a strong candidate gene (DRD2, which encodes the D2 dopamine receptor) disrupts alternative splicing and leads to inappropriate transcription that is associated with cognitive dysfunction. Collectively, these results reinforce the notion that consideration of genetic variants that dysregulate particular mRNA isoforms and understanding the biological consequence of expressing such isoforms is a crucial step in our efforts to understand human behavior and to develop therapeutic interventions for mental disorders.
    • ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS

      Moffat, Jennifer; DE, CHANDRAV (2015)
      The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes primary and recurrent diseases that are preventable with live vaccines and are treatable with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4- yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and mechanism of action in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to LBHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drugdrug interactions were not known. Given its similar structure to brivudine (BVdU), we addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. LBHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by evaluating its activity against related α-herpesviruses and by analyzing resistant VZV viii strains. VZV strains resistant to L-BHDU (L-BHDUR ) were cross-resistant to ACV and BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR virus did not phosphorylate the drug. We also investigated whether addition of nucleosides reversed LBHDU inhibition of VZV in dividing and quiescent HFFs. Excess thymidine and uridine, but not purines, in proportion to L-BHDU restored VZV replication only in dividing cells, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. Like other herpesviruses, VZV infection induced thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool. Some herpesviruses raise dNTP pools by inducing cellular enzymes. However, VZV infection did not increase cellular thymidylate synthase (TS) expression to facilitate viral replication. Furthermore, the active form of L-BHDU did not interfere with cellular metabolism, suggesting a viral target. Further studies are required to identify the target(s) of L-BHDU active form(s).
    • Biological importance of TIMP-2 phosphorylation on MMP-2 activity

      Bourboulia, Dimitra; Bullard, Renee (2016)
      Matrix metalloproteinases (MMPs) are proteolytic enzymes that are secreted from the cell and play an important role in embryonic development and tissue remodeling. In cancer, MMPs are hyperactive, promoting degradation of the ex-tracellular matrix. Enhancement of MMP proteolytic activity allows tumor cells to migrate and invade surrounding tissues, increasing the chance of metastasis. Tissue inhibitor of metalloproteinases (TIMPs) are also known to act extracellu-larly, and are the endogenous inhibitors of MMPs. To inhibit the protease activi-ty of MMPs, the N-terminus of the TIMP protein binds to the catalytic domain of MMP at a ratio of 1:1. Studies from our lab have found that TIMP-2 is phosphor-ylated on three tyrosine residues, and this phosphorylation increases the inter-action with MMP-2. This is the first time that phosphorylation of TIMP-2 has been reported. Fascinatingly, the proto-oncogene tyrosine kinase c-Src was found to phosphorylate TIMP-2. This is significant in that c-Src has not yet been shown to act extracellularly, and there are no details within the current lit-erature describing how this protein may function outside of the cell. In this the-sis, we usedmammalian cells as a model to decipher whether TIMP-2 phosphor-ylation wasable to occur extracellularly,as well as the effect that phosphoryla-tion of TIMP-2 hadon its functionto both inhibit/activate MMP-2. We found that(1) c-Src is able to phosphorylate TIMP-2 extracellularly in conditioned me-vidia; and (2) phosphorylation of TIMP-2 enhances its function of inhibiting MMP-2 proteolytic activity, as well as assisting in the activation of pro-MMP-2. Our results suggest the presence of anovel mechanismin whichphosphoryla-tion of TIMP-2is able to regulate the extracellular environment through en-hanced interaction with MMP-2. The information gained from this research couldlead to development of novel therapies that use phosphorylated TIMP-2 as a means of decreasing cellular migration and invasion with the overall goal of preventing metastasis.
    • Connexin43 and immunity : macrophage phagocytosis, cardiac calcinosis and autoimmune myocarditis

      Steven Taffet; Aaron Glass (2013)
      Connexin43 (Cx43) is a gap junction protein best known for coupling the cytoplasms of cardiac myocytes and allowing the efficient conduction of action potentials throughout the heart. In addition to the heart, Cx43 is also highly expressed in many immune cells and it has been attributed numerous roles in immunity. One such reported role was in macrophage phagocytosis. The first chapter in this dissertation explored the phagocytic activity of cultured and primary murine macrophages from wild type (WT) and Cx43-deleted (Cx43-/-) macrophages. No difference in phagocytic uptake was observed between the two groups using a series of target particles, indicating that Cx43 is dispensable for phagocytosis in macrophages. Given the spectrum of immune functions in which Cx43 has been ascribed a role, we set out to characterize its effect on a model of autoimmune myocarditis (EAM). Using the area of cardiac inflammatory infiltrate as a correlate of disease severity, we observed the progression of the disease to be independent of Cx43 status utilizing WT and Cx43-heterozygous (Cx43+/-) animals as well as radiation chimeric mice reconstituted with cells from donor WT, Cx43+/- and Cx43-/- mice. Although the severity of EAM did not measurably change when induced in animals with differing levels of Cx43 expression, substantial changes to ventricular Cx43 were noted in diseased hearts. Large foci were observed that completely lacked Cx43 immunofluorescence signal. Areas surrounding these foci exhibited disrupted Cx43 patterns such as internalization and lateralization. Similar alterations to Cx43 were also observed in the BALB/cByJ strain of laboratory mice that develop a spontaneous myocarditic disease. To investigate the electrophysiological ramifications of EAM, especially in the context of Cx43+/- mice, ECGs were recorded from animals over the course of EAM. Significant changes to the QRS interval were noted, including prolongation that was only observed in Cx43+/- animals.
    • EVALUATION OF THE ROLE OF THE CENTRAL PAIR IN CHLAMYDOMONAS REINHARDTII FLAGELLAR MOTILITY

      Mitchell, David; Brandon, Smith (2013)
      Cilia and flagella are essential for the function of nearly all eukaryotes. This organelle is made up of nine outer doublet microtubules and two central singlet microtubules to form the canonical (9+2) ciliary structure. Cilia and flagella use this structure, as well as several protein complexes, such as the outer and inner dynein arms, the radial spokes, and the proteins that decorate the central pair to propagate the bending that produces motion. Flagellar motion is highly regulated, and each of these structures is necessary to regulate the dynein arms that generate the motile force. The central pair is one of the least understood of these structures. To date there are two major impediments hindering our understanding of the central pair: a lack of understanding as to how distinct central pair structures work in concert, and a general lack of available central pair mutant strains in the model organism Chlamydomonas reinhardtii. In order to further our understanding of how the central pair functions I have used multiple strategies. Firstly I have used previously characterized central pair mutants to study both structural interactions within the central pair and how the double mutant affects motility regulation. Secondly I provide evidence that a potential central pair mutant, H2, is indeed a central pair mutant and affects the C2b projection. Lastly I will attempt to characterize a new Chlamydomonas mutant, 10B5. Together these analyses will demonstrate that double mutants can have an additive effect on the structure of the central pair, and that double central pair mutants do not appear to suppress one another, but are at least ivepistatic to the most severe phenotype. I will also show evidence that 10B5 is not a central pair mutant, but with further study it may offer new insight into motilityregulation.
    • Expression and Function of Paxillin Genes in Zebrafish: A Role in Skeletal Muscle Development

      Turner, Chris; Amack, Jeffrey; Jacob, Andrew (2017)
      Paxillin is a key component of the Integrin adhesion complex, which regulates cellular signaling events in response to extracellular matrix interactions. Although the roles for Paxillin in cell migration have been extensively studied, less is understood about its role in vertebrate development. Depletion of Paxillin from mouse embryos results in early lethality due to impaired cardiovascular development and function, necessitating the development of alternative vertebrate genetic models for examining the role of Paxillin during embryogenesis. Zebrafish have emerged as an experimental vertebrate model amenable to genetic manipulation. The work compiled herein first characterizes the expression profiles for Paxillin genes in zebrafish, and then describes the embryonic phenotypes observed upon mutation of these genes. The identification of two Paxillin genes in zebrafish, pxnaand pxnb, provided new insight into the evolution of this gene family in the Teleost lineage. Both overlapping and unique expression profiles for these genes during zebrafish embryogenesis were uncovered. While both genes are expressed in developing skeletal muscle, pxnawas restricted to the notochord during earlier stages of embryogenesis and pxnbwas expressed in the developing heart. Targeted mutation of either gene alone did not impair embryonic development, suggesting partial functional redundancy between each gene during embryogenesis. Accordingly, combined mutations in pxnaand pxnbrevealed defects during the development ofseveral embryonic tissues. In particular, skeletal muscle morphogenesis iiiwas perturbed in these double mutant embryos. Further characterization revealed that Paxillin genes in zebrafish serve to regulate embryonic myotome shape and proper extracellular matrix composition during muscle development. The amount of Laminin was reduced, while the abundance of Fibronectin persisted, during myotome morphogenesis in Paxillin double mutant embryos. In addition, a role for cytoskeletal contractility in regulatingsubcellular localization of Paxillin in developing skeletal muscle was established. Defects in the development of the cardiovascular system were also apparent in Paxillin double mutant embryos, and future work will focus on characterizing these in further detail. Altogether, this work provides a new vertebrate model to use for understanding the role of Paxillin during embryonic development, and uncovers an unrecognized role for Paxillin in establishing the extracellular matrix of skeletal muscle.
    • Investigating the Role of Paxillin in Mammary Gland Morphogenesis and Breast Cancer Progression

      Turner, Christopher; Xu, Weiyi (2020)
      Breast cancer is one of the most invasive cancers among women. Understanding the mechanisms contributing to breast cancer progression may identify potential ways to prevent and cure the disease. Meanwhile, mammary gland morphogenesis shares similar mechanisms to allow the ducts to invade and occupy the fat pad. Thus, it is equally important to investigate the normal mammary gland development as breast tumor progression. Paxillin, as a focal adhesion scaffold protein, has previously been implicated in multiple types of cancer cell migration and invasion through its role in cell- ECM signaling. Herein, I utilized a novel paxillin conditional knockout mouse model and paxillin knockout mouse crossed with PyMT breast tumor mouse model to show that paxillin is critical for both mammary gland morphogenesis and breast tumor progression. In Chapter 2, by evaluating the developing mammary gland morphology with immunohistochemistry and the three-dimensional cultured mammary organoids and acini, a critical role of paxillin was shown in facilitating apical-basal polarity formation in the luminal epithelial cells in part, through its control of HDAC6 activity and associated microtubule acetylation. Correct polarization and columnar shape of the epithelial cells potentially contributes to lumen formation and branching of the ducts. Investigation in Chapter 3 highlights a crucial role of paxillin in breast cancer invasion and distant organ metastasis, but did not affect the primary tumor growth rate. Further analysis revealed that paxillin is required for the endocytosis and recycling of E- cadherin, which is important for the maintenance of Adherens junction equilibrium during cancer cell collective migration. This thesis characterizes the roles for paxillin in mammary gland morphogenesis and breast cancer progression and reveals the importance of paxillin-dependent apical trafficking in normal epithelial cells, and E-cadherin trafficking in collective migrating tumor cells. Together, this work highlights a trafficking-dependent mechanism for paxillin during both physiologic and pathologic processes in the mammary gland.
    • Laminins regulate retinal angiogenesis

      Brunken, William J.; Biswas,Saptarshi (2017)
      Vascular pathologies are the leading causes of acquired blindness in the developed world. While many studies sought to unravel cell-intrinsic and growth factor-mediated regulations of angiogenesis, it is only recently that the role of the basement membrane (BM) components in angiogenesis began to be explored. Several diseases with ocular manifestations are known to alter vascular BM compositions. Therefore, a detailed knowledge of the BM-mediated signals that regulate angiogenesis is of great importance. Laminins, a critical component of the BM, have been shown to regulate several aspects of angiogenesis in the retina. Our laboratory previously demonstrated that the laminin composition of the inner limiting membrane (ILM) regulates astrocyte migration, and consequently vascular expansion along the retinal surface. Here, I examined the role of γ3- and β2-containing laminins in two specific aspects of angiogenesis: 1) vascular branching and endothelial cell proliferation in the nascent vascular plexus, and 2) arterial morphogenesis in the remodeling zone. Results presented in Chapter 2 and Appendix 1 demonstrate that laminin composition of the BM is a critical regulator of microglial recruitment to the growing nascent plexus, where microglia facilitate vascular branching. Furthermore, microglia interact with the astrocyte-derived layer of the vascular BM, and that this interaction regulates iii microglial activation. The activation state of microglia, in turn, regulates endothelial cell proliferation. Results presented in Chapter 3 and Appendix 2 demonstrate that vascular BM laminins are critical regulators of arterial morphogenesis. Specifically, my results reveal a novel mechanism where γ3- containing laminins signal through dystroglycan to induce Dll4/Notch signaling in arterial endothelial cells, regulating proper arterial morphogenesis. Finally, in Appendix 3, I examined the coordinated expression of different laminin chains in the vascular BM. My preliminary results suggest that expressions of laminin α2-, α5- and γ3-chains in the retinal vascular BM are coordinately regulated with the expression of laminin β2-chain. In conclusion, this study sheds light on hitherto unexplored mechanisms by which BM laminins regulate retinal vascular development.
    • Mechanisms of aseptic loosening in total knee arthroplasty

      Cyndari, Karen (2017)
      Introduction: Cemented Total Knee Arthroplasty (TKA) is the gold standard of care for end-stage, multi-etiologic arthritis. While the longevity of these devices may now reach or even surpass 15 years in service, a minority (~10%) will fail prematurely due to a process called aseptic loosening. Historically, this process has been attributed to an inflammatory reaction against wear debris from the TKA polyethylene (PE) insert. However, we have previously estimated supraphysiologic fluid shear stress (FSS) (exceeding 900 Pa) at the cement-bone interface of cemented joint replacements, and examined this as a possible alternative cause of increased osteoclast activity. Methods: We analyzed the cement-bone interlock of tibial and femoral components from en-bloc, postmortem-retrieved, non-revised TKAs to explore the process of loss of fixation, prior to any clinical loosening. For the tibial components, we used a novel protocol wherein whole undecalcified bone+PMMA cement segments from the proximal tibia were embedded in Spurr’s resin and thinly sectioned. Polarized light microscopy was used to identify and quantify co-located PE debris. Using a novel bioreactor developed by our lab called the Multi-Well Fluid Loading (MFL) System, we examined static, subphysiologic, physiologic, and supraphysiologic FSS on RAW 264.7 osteoclast activity and morphology, with and without PE particle treatment. Results: We found no association been the amount or presence of PE debris and the amount or location of loss of interlock in retrieved TKAs. FSS up to 17 Pa increased the ability of osteoclasts to resorb mineral, and FSS up to 4.4 Pa induced the formation of larger osteoclasts. FSS and fluid shear rate interacted together to increase the area of actin rings, while PE treatment increased the number of actin rings and TRAP production. FSS up to 4.4 Pa decreased expression of Ctsk and Il1a, but PE co-treatment abolished this effect. Conclusions: These results indicate there may be alternative factors leading to aseptic loosening apart from PE debris. We demonstrated that osteoclasts are mechanosensitive and able to adjust activity, morphology, and gene expression based on FSS. Further, PE interferes with osteoclast gene downregulation in response to FSS, indicating PE could be a potentiator of osteoclast activity or differentiation.
    • MECHANISMS OF WISKOTT ALDRICH SYNDROME PROTEIN WSP1 POSITIONING AND REGULATION AT SITES OF ENDOCYTOSIS IN S. POMBE

      Sirotkin, Vladimir; Macquarrie, Cameron Dale (2020-12-30)
      Branched actin networks nucleated by the Arp2/3 complex provide force needed to carry out endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including Wiskott-Aldrich Syndrome protein WASp. The WASp Interacting Protein WIP binds WASp, protecting it from degradation. Humans with mutations disrupting this interaction develop a serious immune disorder, Wiskott-Aldrich Syndrome. However, in the fission yeast S. pombe WASp homolog Wsp1 remains stable in the absence of WIP homolog Vrp1, providing an ideal environment to study additional WASp and WIP functions. In fission yeast, Wsp1, Vrp1, and the class-1 myosin Myo1 localize to sites of endocytosis, known as actin patches. Wsp1 and Myo1 play an important role in activating the Arp2/3 complex and initiating the actin network needed to internalize endocytic vesicles. S. pombe endocytosis is a rapid, reproducible event involving over 40 proteins. While several of these proteins are throught to regulate branched actin assembly, many still have poorly defined functions. Importantly, how WASp proteins are regulated at sites of endocytosis remains unclear. The following studies explore mechanisms of Wsp1 regulation using quantitative live cell imaging. In Chapter 2, we observed Wsp1, Vrp1, and Myo1 forming a transient complex near the membrane, positioning branched filaments in a way that optimizes force generation. In Chapter 3, we explored the role of WIP homolog Vrp1 in actin assembly and discovered the Vrp1-Wsp1 interaction is essential for Wsp1-mediated branched actin assembly. In Chapter 4, we examined how a separate module of endocytic coat proteins contributes to actin patch assembly and discovered the coat protein Sla1 inhibits Wsp1 NPF activity, after the endocytic vesicle begins to internalize. In Chapter 5, we examined how Wsp1 and Myo1 impact additional endocytic modules and discovered Wsp1 plays an important role in expediating endocytosis and Myo1 contributes to the localization of several proteins. Lastly, in Chapter 6, we observed that blocking the fastgrowing end of actin filaments does not impact actin assembly in patches. These studies provide key insight into how WASp family proteins are regulated in vivo.
    • Role of RPTPzeta/phosphacan in the neural extracellular matrix

      Matthews, Rick; Eill, Geoffrey (2020)
      The mammalian brain is the most structurally and functionally complex system in biology. In order to carry out diverse functions such as thought and cognition, neurons in the brain must properly differentiate, make millions of functional interconnections, and incredibly be able to maintain those interconnections while retaining plasticity and the ability to learn throughout its lifetime. In order for neurons to carry out these complex functions, they must have an intricate relationshipwith their extracellular environment, which provides numerous molecules, such as growth factors and neurotransmitters, and physical cues to initiate critical downstream signaling cascades. In the central nervous system (CNS), the neural extracellular matrix (ECM) largely organizes this extracellular environment, and as such, is implicated in a multitude of neuronal functions. Not only can it serve as a physical barrier, the neural ECM is shown to regulate neuronal cell differentiation, migration, synaptogenesis, and maintain the mature state by restricting plasticity and neurite outgrowth. Of particular import, a subset of the neural ECM, the perineuronal net (PNN), is implicated to regulate neuronal plasticity in the CNS. PNNs were historically believed to be critical for restricting experience dependent plasticity in the brain but more recently shown to regulate several forms of learning and memory, in addition to multiple neurological diseases. However, despite these implicated functions, PNNs comprise only a fraction of the total ECM in the CNS. A majority of the neural ECM is derived from the diffuse ECM, a structure that is ubiquitously expressed throughout the CNS. As the diffuse ECM is very similar in molecular composition and structure to PNNs, it is difficult to specifically target PNNs for functional and mechanistic studies.As such, a better toolset is needed to differentiate the contributions ivbetween PNNs and the diffuse ECM in regulating plasticity and learning and memory. To develop thistoolset, there is a great need to better understand PNN molecular composition and structure. Therefore, the purpose of this work was to provide critical insight into the molecular composition and structure of PNNs soas to better understand its function in the CNS. In Chapter 2, using multiple ECM genetic knockout models, we show compelling evidence of an additional PNN anchor that importantlylays the groundworkforfuture functional studies. Specifically, we suggestchondroitin sulfate proteoglycan (CSPG) phosphacan, secreted isoform of receptor protein tyrosine phosphatase zeta (RPTPζ), iscritical for PNN structure as it partially anchors PNNs to the neuronal surface through cooperation with tenascin-R. Additionally, as the neural ECM is involved in numerous neurological diseases, in Chapter 3, we investigated the function of major ECM component, RPTPζ, in a group of O-mannosyl related congenital muscular dystrophy with associated brain abnormalities (CMD). Our data suggest a possible role of RPTPζ in proper cortical lamination in a CMD mouse model. Interestingly, we found evidence of a novel O-mannosyl substrate in the developing brain that could critically contribute to the underlying deficits of CMD. In conclusion, the neural ECM, once previously disregarded in the field, is becoming a novel source to understand mature mammalian brain function and disease, but more work is needed to better differentiate the specific roles of its substructures.
    • Specific mutations in the α and ß subunits of the Kluyveromyces lactis F1-ATPase enhance ATP hydrolysis in the absence of the central γ-rotor

      Xin Jie Chen; Thuy La (2013)
      In eukaryotic cells, the mitochondria are vital organelles which are required for cell viability. Mitochondrial stresses such as oxidative stress, loss of membrane potential or loss of mitochondrial DNA are considered extreme and are associated with many neurodegenerative diseases and aging. The mitochondrial FoF1-synthase, where the majority of cellular ATP is synthesized, is composed of one inner membrane bound Fo domain and a water soluble F1 domain in the mitochondrial matrix. F1 contains the hexameric α3β3core and the centrally located γ subunit. The γ subunit is believed to play a key role in inducing conformational changes while rotating within the α3β3 core during ATP hydrolysis/synthesis. Previous studies have shown that the α3β3 core alone from the Thermophilic bacterium PS3 has a detectable hydrolyzing activity. In recent years, evidence of the rotary catalysis of Thermophilic Bacillus sp. PS3 F1-ATPase without its rotor - subunit γ - was shown using high-speed atomic force microscopy[1]. Moreover, previous study undertaken in our lab had utilized a unique genetic screen that allowed the identification of two specific mutations in the α and β subunits in the aerobic yeast Kluyveromyces lactis that stimulate ATP hydrolysis by the mitochondrial F1-ATPase in the absence of γ. This allows cells to survive upon the loss of mitochondrial DNA. In current work, we confirmed that the αF446I and βG419D mutations on the DELSEED loop are sufficient to allow ρ0 cells to survive in the absence of γ. Biochemical experiments showed that the γ -less F1-ATPase can be assembled to actively hydrolyze iv ATP in vivo, but this activity becomes extremely labile in vitro. These studies give insights into the catalytic mechanism of the α3β3 subcomplex and help to better understand the evolutionary origin of the mitochondrial F1-ATPase.
    • Structural and functional characterization of the V-ATPase membrane sector

      Couoh-Carde, Sergio J. (2017)
      The vacuolar ATPase (V1VO-ATPase, V-ATPase) is a H+-pump involved in the acidification of organelles in eukaryotes. Under certain physiological conditions, the VATPase disassociates into an inactive soluble ATPase sector (V1) and a membrane sector (VO) that is impermeable to protons. Due to the lack of detailed structural and functional information, the auto-inhibition mechanism of VO is not well understood. Although the V-ATPase shares a similar structure and rotary catalysis mechanism with the F- and AATPases, V-ATPase’s increased structural complexity and unique mode of regulation suggest other functions beyond its canonical proton pumping. We purified Vo and Vo sub-complexes for structural and functional characterization. First, our ~18 Å cryo-EM model of Vo suggests that c-ring (c8c’c’’) is partially surrounded by the C-terminal membrane integral portion of subunit a (aCT). On the other hand, the soluble N-terminal portion of subunit a (aNT) interacts with subunit d that sits atop of the c-ring. Selective removal of subunit d (VoΔd) did not allow passive proton translocation. Second, the c-ring was isolated and its X-ray crystal structure was solved at ~4 Å resolution. Two c-rings interact to form a gap-junction like structure. The presence of c’’ disrupts the intrinsic and global symmetry of the c8c’ sub-complex, constituting a kinetic barrier during c-ring axial rotation. Third, we discovered that c-ring can act as a large-conductance ion-channel independently from its canonical function in proton pumping. Our biophysical, biochemical, and functional data suggest that exquisite kinetic barriers play a primary role in the auto-inhibition of Vo, and that Vo may have noncanonical functions in intercellular communication.
    • THE ROLE OF DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 IN DISEASE PATHOGENESIS

      King, Christine; Endy, Timothy; Barbachano-Guerrero, Arturo (2020)
      Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage and an over reactive inflammatory response. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. Mast cells (MC) are potent cells of the innate immune system that play an important role in EC biology and inflammatory responses. DENV encodes 10 proteins; with only one, the non-structural protein 1 (NS1), secreted from infected cells and accumulating in the blood of patients.NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Using a complementary array of in vitroassays and disease relevant ECs and MCs, we described the possible roles for NS1 in dengue disease pathogenesis. Using microscopy and immunoblotting we observed that ECs internalize NS1 into endosomes, where it accumulates and is degraded overtime. Transcriptome and pathway analysis defined changes in global gene expression in ECs that are associated with cell dysfunction. We observed that NS1 induced an increase in multicellular rearrangements and a decrease in barrier function in ECs. We demonstrated that NS1-dependent activation of the p38 MAPK pathway controls the changes in EC permeability in vitro. Further, we discovered iiithat ECs and MCs respond to NS1 by secreting a specific array of proinflammatory cytokines and chemokines that may contribute to the cytokine storm in dengue disease. Finally, we found that NS1 internalization can mediate the uptake of bound antibodies into ECs. Together, these results suggest a vasoactive and proinflammatory role for DENV NS1 that may participate in the development of severe symptoms in dengue disease. The observed functions of NS1 could lead to the discovery of new therapeutic targets in dengue disease.