Medicine and Health Sciences Commons^™

Kras Inhibitor That Simultaneously Inhibits Nucleotide Exchange Activity And Effector Engagement, Cynthia V Pagba, Amit K Gupta, Ali K Naji, Dharini Van Der Hoeven, Kelly Churion, Xiaowen Liang, Jacob Jakubec, Magnus Hook, Yan Zuo, Marisela Martinez De Kraatz, Jeffrey A Frost, Alemayehu A Gorfe

Journal Articles

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and …

In Silico Identification Of A Β2-Adrenoceptor Allosteric Site That Selectively Augments Canonical Β2ar-Gs Signaling And Function, Sushrut D Shah, Christoffer Lind, Francesco De Pascali, Raymond B Penn, Alexander D Mackerell, Deepak A Deshpande

Department of Biochemistry and Molecular Biology Faculty Papers

Activation of β2-adrenoceptors (β2ARs) causes airway smooth muscle (ASM) relaxation and bronchodilation, and β2AR agonists (β-agonists) are front-line treatments for asthma and other obstructive lung diseases. However, the therapeutic efficacy of β-agonists is limited by agonist-induced β2AR desensitization and noncanonical β2AR signaling involving β-arrestin that is shown to promote asthma pathophysiology. Accordingly, we undertook the identification of an allosteric site on β2AR that could modulate the activity of β-agonists to overcome these limitations. We employed the site identification by ligand competitive saturation (SILCS) computational method to comprehensively map the entire 3D structure of in silico-generated β2AR intermediate conformations and identified …

Unilateral ‘Crazy-Paving’ Pattern: An Exceptional Presentation Of Squamous Cell Carcinoma Of The Lung, Sumeera Banday, Shamim U Khan, Sabina Khan, Surinderpal S Anand, Ashok Shah

Journal Articles

The 'crazy-paving' pattern on high resolution computed tomography (HRCT) is a distinct imaging appearance that is still considered to be a radiological hallmark of pulmonary alveolar proteinosis. However, since its first description about three decades ago, more than 40 different clinical entities presenting as 'crazy-paving' patterns have been documented. This rather remarkable but uncommon imaging appearance is now considered to be a non-specific manifestation. A 62-year-old male referred for evaluation of productive cough, breathlessness and fever presented with 'crazy-paving' pattern on HRCT. Endobronchial biopsy done on presentation was consistent with the diagnosis of squamous cell carcinoma. This report highlights this …

Right Ventricular Energetic Biomarkers From 4d Flow Cmr Are Associated With Exertional Capacity In Pulmonary Arterial Hypertension., Xiaodan Zhao, Shuang Leng, Ru-San Tan, Ping Chai, Tee Joo Yeo, Jennifer Ann Bryant, Lynette L S Teo, Marielle V Fortier, Wen Ruan, Ting Ting Low, Ching Ching Ong, Shuo Zhang, Rob J Van Der Geest, John C Allen, Marina Hughes, Pankaj Garg, Teng Hong Tan, James W Yip, Ju Le Tan, Liang Zhong

Journal Articles

BACKGROUND: Cardiovascular magnetic resonance (CMR) offers comprehensive right ventricular (RV) evaluation in pulmonary arterial hypertension (PAH). Emerging four-dimensional (4D) flow CMR allows visualization and quantification of intracardiac flow components and calculation of phasic blood kinetic energy (KE) parameters but it is unknown whether these parameters are associated with cardiopulmonary exercise test (CPET)-assessed exercise capacity, which is a surrogate measure of survival in PAH. We compared 4D flow CMR parameters in PAH with healthy controls, and investigated the association of these parameters with RV remodelling, RV functional and CPET outcomes.

METHODS: PAH patients and healthy controls from two centers were prospectively …

Terminase Subunits From The Pseudomonas-Phage E217, Ravi K Lokareddy, Chun-Feng David Hou, Steven G Doll, Fenglin Li, Richard E Gillilan, Francesca Forti, David S Horner, Federica Briani, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed …

The Detection Of Periodic Reemergence Events Of Sars-Cov-2 Delta Strain In Communities Dominated By Omicron, Claire E. Westcott, Kevin J. Sokoloski, Eric C. Rouchka, Julia H. Chariker, Rochelle H. Holm, Ray A. Yeager, Joseph B. Moore Iv, Erin M. Elliott, Daymond Talley, Aruni Bhatnagar

Faculty Scholarship

Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community …

Integrating Genomic Information With Tumor-Immune Microenvironment In Triple-Negative Breast Cancer, David Otohinoyi, Aditi Kuchi, Jiande Wu, Chindo Hicks

School of Graduate Studies Faculty Publications

Background: the development and progression of triple-negative breast cancer (TNBC) is driven by somatic driver mutations and the tumor-immune microenvironment. To date, data on somatic mutations has not been leveraged and integrated with information on the immune microenvironment to elucidate the possible oncogenic interactions and their potential effects on clinical outcomes. Here, we investigated possible oncogenic interactions between somatic mutations and the tumor-immune microenvironment, and their correlation with patient survival in TNBC. Methods: We performed analysis combining data on 7,875 somatic mutated genes with information on 1,751 immune-modulated genes, using gene-expression data as the intermediate phenotype, and correlated the resulting …

Structure Of The Pre-Mrna Leakage 39-Kda Protein Reveals A Single Domain Of Integrated Zf-C3hc And Rsm1 Modules, Hideharu Hashimoto, Daniel H. Ramirez, Ophélie Lautier, Natalie Pawlak, Günter Blobel, Benoît Palancade, Erik W. Debler

Department of Biochemistry and Molecular Biology Faculty Papers

In Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 Å resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP …

Effects Of Cannabinoids On Ligand-Gated Ion Channels, Murat Oz, Keun-Hang Susan Yang, Mohamed Omer Mahgoub

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Phytocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol, endocannabinoids such as N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, and synthetic cannabinoids such as CP47,497 and JWH-018 constitute major groups of structurally diverse cannabinoids. Along with these cannabinoids, CB1 and CB2 cannabinoid receptors and enzymes involved in synthesis and degradation of endocannabinoids comprise the major components of the cannabinoid system. Although, cannabinoid receptors are known to be involved in anti-convulsant, anti-nociceptive, anti-psychotic, anti-emetic, and anti-oxidant effects of cannabinoids, in recent years, an increasing number of studies suggest that, at pharmacologically relevant concentrations, these compounds interact with several molecular targets including G-protein coupled receptors, ion …

Viral Small Terminase: A Divergent Structural Framework For A Conserved Biological Function., Ravi K. Lokareddy, Chun-Feng David Hou, Fenglin Li, Ruoyu Yang, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a …

The Role Of Ubiquitination In Spinal And Bulbar Muscular Atrophy, Medha Sengupta, Anna Pluciennik, Diane E. Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative and neuromuscular genetic disease caused by the expansion of a polyglutamine-encoding CAG tract in the androgen receptor (AR) gene. The AR is an important transcriptional regulator of the nuclear hormone receptor superfamily; its levels are regulated in many ways including by ubiquitin-dependent degradation. Ubiquitination is a post-translational modification (PTM) which plays a key role in both AR transcriptional activity and its degradation. Moreover, the ubiquitin-proteasome system (UPS) is a fundamental component of cellular functioning and has been implicated in diseases of protein misfolding and aggregation, including polyglutamine (polyQ) repeat expansion diseases …

Young Transposable Elements Rewired Gene Regulatory Networks In Human And Chimpanzee Hippocampal Intermediate Progenitors, Sruti Patoori, Samantha M Barnada, Christopher Large, John I Murray, Marco Trizzino

Department of Biochemistry and Molecular Biology Faculty Papers

The hippocampus is associated with essential brain functions, such as learning and memory. Human hippocampal volume is significantly greater than expected compared with that of non-human apes, suggesting a recent expansion. Intermediate progenitors, which are able to undergo multiple rounds of proliferative division before a final neurogenic division, may have played a role in evolutionary hippocampal expansion. To investigate the evolution of gene regulatory networks underpinning hippocampal neurogenesis in apes, we leveraged the differentiation of human and chimpanzee induced pluripotent stem cells into TBR2 (or EOMES)-positive hippocampal intermediate progenitor cells (hpIPCs). We found that the gene networks active in hpIPCs …

Enhanced Membrane Binding Of Oncogenic G Protein Αqq209l Confers Resistance To Inhibitor Ym-254890, Clinita E. Randolph, Morgan B. Dwyer, Jenna L. Aumiller, Alethia J. Dixon, Asuka Inoue, Patrick Osei-Owusu, Philip B. Wedegaertner

Department of Biochemistry and Molecular Biology Faculty Papers

Heterotrimeric G proteins couple activated G protein-coupled receptors (GPCRs) to intracellular signaling pathways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the αqQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT αq and GPCR-independent αqQ209L. Although YM inhibits WT αq by binding to αq-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular αqQ209L remains to be fully understood. Here, we show that YM promotes a subcellular redistribution of αqQ209L from the plasma membrane (PM) to the cytoplasm. …

Isc10, An Inhibitor Of The Smk1 Mapk, Prevents Activation Loop Autophosphorylation And Substrate Phosphorylation Through Separate Mechanisms, Abhimannyu Rimal, Thomas M Swayne, Zeal P Kamdar, Madison A Tewey, Edward Winter

Department of Biochemistry and Molecular Biology Faculty Papers

Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system …

The Pro-Fibrotic Response To Lens Injury Is Signaled In A Pi3k Isoform-Specific Manner, A. Sue Menko, Janice L. Walker

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as …

G Protein-Coupled Receptor Kinase 6 (Grk6) Regulates Insulin Processing And Secretion Via Effects On Proinsulin Conversion To Insulin, Matthew J Varney, Wouter Steyaert, Paul J Coucke, Joris R Delanghe, David E Uehling, Babu Joseph, Richard Marcellus, Rima Al-Awar, Jeffrey L Benovic

Department of Biochemistry and Molecular Biology Faculty Papers

Recent studies identified a missense mutation in the gene coding for G protein-coupled receptor kinase 6 (GRK6) that segregates with type 2 diabetes (T2D). To better understand how GRK6 might be involved in T2D, we used pharmacological inhibition and genetic knockdown in the mouse β-cell line, MIN6, to determine whether GRK6 regulates insulin dynamics. We show inhibition of GRK5 and GRK6 increased insulin secretion but reduced insulin processing while GRK6 knockdown revealed these same processing defects with reduced levels of cellular insulin. GRK6 knockdown cells also had attenuated insulin secretion but enhanced proinsulin secretion consistent with decreased processing. In support …

Subtype-Selective Positive Modulation Of KCa2.3 Channels Increases Cilia Length, Young-Woo Nam, Rajasekharreddy Pala, Naglaa Salem El-Sayed, Denisse Laren-Henriquez, Farideh Amirrad, Grace Yang, Mohammad Asikur Rahman, Razan Orfali, Myles Downey, Keykavous Parang, Surya M. Nauli, Miao Zhang

Pharmacy Faculty Articles and Research

Small-conductance Ca²⁺-activated potassium (K_Ca2.x) channels are gated exclusively by intracellular Ca²⁺. The activation of K_Ca2.3 channels induces hyperpolarization, which augments Ca²⁺ signaling in endothelial cells. Cilia are specialized Ca²⁺ signaling compartments. Here, we identified compound 4 that potentiates human K_Ca2.3 channels selectively. The subtype selectivity of compound 4 for human K_Ca2.3 over rat K_Ca2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of K_Ca2.3 channels by compound 4 increased flow-induced Ca²⁺ signaling and cilia length, while negative …

Glucose Uptake By Glut1 In Photoreceptors Is Essential For Outer Segment Renewal And Rod Photoreceptor Survival, Lauren L. Daniele, John Y.S. Han, Ivy S Samuels, Ravikiran Komirisetty, Nikhil Mehta, Jessica L Mccord, Minzhong Yu, Yekai Wang, Kathleen Boesze-Battaglia, Brent A Bell, Jianhai Du, Neal S Peachey, Nancy J. Philp

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or …

Mettl14-Mediated Epitranscriptome Modification Of Mn1 Mrna Promote Tumorigenicity And All-Trans-Retinoic Acid Resistance In Osteosarcoma, Hong-Bo Li, Gang Huang, Jian Tu, Dong-Ming Lv, Qing-Lin Jin, Jun-Kai Chen, Yu-Tong Zou, Dung-Fang Lee, Jing-Nan Shen, Xian-Biao Xie

Journal Articles

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor in adolescents. The molecular mechanism behind OS progression and metastasis remains poorly understood, which limits the effectiveness of current therapies. RNA N

METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS), dot blotting, and colorimetric ELISA were used to detect m

FINDINGS: We observed the abundance of m

INTERPRETATION: Our study revealed that METTL14 contributes to OS progression and ATRA resistance as an m

FUNDING: This work was supported by the National Natural Science Foundation of China (Grants 81972510 and 81772864).

Synthesis And Evaluation Of Anti-Hiv Activity Of Mono- And Di-Substituted Phosphonamidate Conjugates Of Tenofovir, Aaminat Qureshi, Louise A. Ouattara, Naglaa Salem El-Sayed, Amita Verma, Gustavo F. Doncel, Muhammad Iqbal Choudhary, Hina Siddiqui, Keykavous Parang

Pharmacy Faculty Articles and Research

The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several …

Integrating Conformational Dynamics And Perturbation-Based Network Modeling For Mutational Profiling Of Binding And Allostery In The Sars-Cov-2 Spike Variant Complexes With Antibodies: Balancing Local And Global Determinants Of Mutational Escape Mechanisms, Gennady M. Verkhivker, Steve Agajanian, Ryan Kassab, Keerthi Krishnan

Mathematics, Physics, and Computer Science Faculty Articles and Research

n this study, we combined all-atom MD simulations, the ensemble-based mutational scanning of protein stability and binding, and perturbation-based network profiling of allosteric interactions in the SARS-CoV-2 spike complexes with a panel of cross-reactive and ultra-potent single antibodies (B1-182.1 and A23-58.1) as well as antibody combinations (A19-61.1/B1-182.1 and A19-46.1/B1-182.1). Using this approach, we quantify the local and global effects of mutations in the complexes, identify protein stability centers, characterize binding energy hotspots, and predict the allosteric control points of long-range interactions and communications. Conformational dynamics and distance fluctuation analysis revealed the antibody-specific signatures of protein stability and flexibility of the …

Neuromuscular Junction Pathology Is Correlated With Differential Motor Unit Vulnerability In Spinal And Bulbar Muscular Atrophy, Elana Molotsky, Y Liu, Andrew P Lieberman, Diane E Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, neuromuscular neurodegenerative disease for which there is no cure. The disease is characterized by a selective decrease in fast-muscle power (e.g., tongue pressure, grip strength) accompanied by a selective loss of fast-twitch muscle fibers. However, the relationship between neuromuscular junction (NMJ) pathology and fast-twitch motor unit vulnerability has yet to be explored. In this study, we used a cross-model comparison of two mouse models of SBMA to evaluate neuromuscular junction pathology, glycolytic-to-oxidative fiber-type switching, and cytoskeletal alterations in pre- and postsynaptic termini of tibialis anterior (TA), gastrocnemius, and soleus hindlimb muscles. …

Regulating Phase Transition In Neurodegenerative Diseases By Nuclear Import Receptors, Amandeep Girdhar, Lin Guo

Department of Biochemistry and Molecular Biology Faculty Papers

RNA-binding proteins (RBPs) with a low-complexity prion-like domain (PLD) can undergo aberrant phase transitions and have been implicated in neurodegenerative diseases such as ALS and FTD. Several nuclear RBPs mislocalize to cytoplasmic inclusions in disease conditions. Impairment in nucleocytoplasmic transport is another major event observed in ageing and in neurodegenerative disorders. Nuclear import receptors (NIRs) regulate the nucleocytoplasmic transport of different RBPs bearing a nuclear localization signal by restoring their nuclear localization. NIRs can also specifically dissolve or prevent the aggregation and liquid–liquid phase separation of wild-type or disease-linked mutant RBPs, due to their chaperoning activity. This review focuses on …

Kalium Channelrhodopsins Are Natural Light-Gated Potassium Channels That Mediate Optogenetic Inhibition, Elena G Govorunova, Yueyang Gou, Oleg A Sineshchekov, Hai Li, Xiaoyu Lu, Yumei Wang, Leonid S Brown, François St-Pierre, Mingshan Xue, John L Spudich

Journal Articles

Channelrhodopsins are used widely for optical control of neurons, in which they generate photoinduced proton, sodium or chloride influx. Potassium (K+) is central to neuron electrophysiology, yet no natural K+-selective light-gated channel has been identified. Here, we report kalium channelrhodopsins (KCRs) from Hyphochytrium catenoides. Previously known gated potassium channels are mainly ligand- or voltage-gated and share a conserved K+-selectivity filter. KCRs differ in that they are light-gated and have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 ms following photoactivation. The permeability ratio PK/PNa of …

An Rtn4/Nogo-A-Interacting Micropeptide Modulates Synaptic Plasticity With Age, S. Kragness, Z. Clark, A. Mullin, J. Guidry, L. R. Earls

School of Medicine Faculty Publications

Micropeptides, encoded from small open reading frames of 300 nucleotides or less, are hidden throughout mammalian genomes, though few functional studies of micropeptides in the brain are published. Here, we describe a micropeptide known as the Plasticity–Associated Neural Transcript Short (Pants), located in the 22q11.2 region of the human genome, the microdeletion of which conveys a high risk for schizophrenia. Our data show that Pants is upregulated in early adulthood in the mossy fiber circuit of the hippocampus, where it exerts a powerful negative effect on long-term potentiation (LTP). Further, we find that Pants is secreted from neurons, where it …

Recognition Of The Tdp-43 Nuclear Localization Signal By Importin Α1/Β, Steven G Doll, Hamed Meshkin, Alexander J Bryer, Fenglin Li, Ying-Hui Ko, Ravi K Lokareddy, Richard E Gillilan, Kushol Gupta, Juan R Perilla, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Cytoplasmic mislocalization of the TAR-DNA binding protein of 43 kDa (TDP-43) leads to large, insoluble aggregates that are a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we study how importin α1/β recognizes TDP-43 bipartite nuclear localization signal (NLS). We find that the NLS makes extensive contacts with importin α1, especially at the minor NLS-binding site. NLS binding results in steric clashes with the C terminus of importin α1 that disrupts the TDP-43 N-terminal domain (NTD) dimerization interface. A putative phosphorylation site in the proximity of TDP-43 R83 at the minor NLS site destabilizes binding to importins by reducing …

Phosphatidylserine And Phosphatidylethanolamine Asymmetry Have A Negligible Effect On The Global Structure, Dynamics, And Interactions Of The Kras Lipid Anchor, Mussie K Araya, Alemayehu A Gorfe

Journal Articles

The intrinsically disordered C-terminus of the prominent oncogenic protein KRAS-4B (KRAS) selectively interacts and clusters with phosphatidylserine (PS) lipids in the plasma membrane (PM). This 11-residue segment, called tK, contains a polybasic domain (PBD) of six contiguous lysine residues and a farnesylated cysteine. Previous molecular dynamics (MD) simulation studies of tK in phosphatidylcholine (PC)/PS bilayers have suggested that backbone conformational dynamics modulate tK-PS interactions. These simulations have been conducted in symmetric membranes whereas the PM is compositionally asymmetric, with the inner leaflet, where KRAS is localized, being enriched with PS and phosphatidylethanolamine (PE) lipids. To examine if bilayer asymmetry affects …

Channelopathy Of Small- And Intermediate-Conductance Ca2+-Activated K+ Channels, Young-Woo Nam, Miles Downey, Mohammad Asikur Rahman, Meng Cui, Miao Zhang

Pharmacy Faculty Articles and Research

Small- and intermediate-conductance Ca²⁺-activated K⁺ (K_Ca2.x/K_Ca3.1 also called SK/IK) channels are gated exclusively by intracellular Ca²⁺. The Ca²⁺ binding protein calmodulin confers sub-micromolar Ca²⁺ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca²⁺-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for K_Ca2.1 (SK1), KCNN2 for K_Ca2.2 (SK2), KCNN3 for K …

Genomic Features Underlie The Co-Option Of Sva Transposons As Cis-Regulatory Elements In Human Pluripotent Stem Cells, Samantha M Barnada, Andrew Isopi, Daniela Tejada-Martinez, Clément Goubert, Sruti Patoori, Luca Pagliaroli, Mason Tracewell, Marco Trizzino

Department of Biochemistry and Molecular Biology Faculty Papers

Domestication of transposable elements (TEs) into functional cis-regulatory elements is a widespread phenomenon. However, the mechanisms behind why some TEs are co-opted as functional enhancers while others are not are underappreciated. SINE-VNTR-Alus (SVAs) are the youngest group of transposons in the human genome, where ~3,700 copies are annotated, nearly half of which are human-specific. Many studies indicate that SVAs are among the most frequently co-opted TEs in human gene regulation, but the mechanisms underlying such processes have not yet been thoroughly investigated. Here, we leveraged CRISPR-interference (CRISPRi), computational and functional genomics to elucidate the genomic features that underlie SVA domestication …

Lysosomal Zn 2+ Release Triggers Rapid, Mitochondria-Mediated, Non-Apoptotic Cell Death In Metastatic Melanoma, Wanlu Du, Mingxue Gu, Meiqin Hu, Timothy Nold, Prateeksunder Pinchi, Wei Chen, Michael Ryan, Ahmed Bannaga, Haoxing Xu

Medical Student Research Symposium

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in …