Medical Sciences Commons^™

Gasdermins In Apoptosis: New Players In An Old Game., Corey Rogers, Emad S. Alnemri

Department of Biochemistry and Molecular Biology Faculty Papers

Apoptosis is a form of programmed cell death (PCD) that plays critical physiological roles in removing superfluous or dangerous cell populations that are unneeded or threatening to the health of the host organism. Although the molecular pathways leading to activation of the apoptotic program have been extensively studied and characterized starting in the 1970s, new evidence suggests that members of the gasdermin superfamily are novel pore-forming proteins that augment apoptosis by permeabilizing the mitochondria and participate in the final stages of the apoptotic program by inducing secondary necrosis/pyroptosis. These findings may explain outstanding questions in the field such as why …

Allele-Specific Rna Interference Prevents Neuropathy In Charcot-Marie-Tooth Disease Type 2d Mouse Models., Kathryn H Morelli, Laurie B Griffin, Nettie K Pyne, Lindsay M Wallace, Allison M Fowler, Stephanie N Oprescu, Ryuichi Takase, Na Wei, Rebecca Meyer-Schuman, Dattatreya Mellacheruvu, Jacob O Kitzman, Samuel G Kocen, Timothy J Hines, Emily L Spaulding, James R Lupski, Alexey Nesvizhskii, Pedro Mancias, Ian J Butler, Xiang-Lei Yang, Ya-Ming Hou, Anthony Antonellis, Scott Q Harper, Robert W Burgess

Department of Biochemistry and Molecular Biology Faculty Papers

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation …

Characterization Of Resistance To A Potent D-Peptide Hiv Entry Inhibitor., Amanda R Smith, Matthew T Weinstock, Amanda E Siglin, Frank G Whitby, J Nicholas Francis, Christopher P Hill, Debra M Eckert, Michael J Root, Michael S Kay

Department of Biochemistry and Molecular Biology Faculty Papers

BACKGROUND: PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely …

Structural And Functional Analysis Of Parameters Governing Tankyrase-1 Interaction With Telomeric Repeat-Binding Factor 1 And Gdp-Mannose 4,6-Dehydratase., Travis Eisemann, Marie-France Langelier, John M. Pascal

Department of Biochemistry and Molecular Biology Faculty Papers

Human tankyrase-1 (TNKS) is a member of the poly(ADPribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: The PARylated partner …

Heme And Hemoglobin Utilization By Mycobacterium Tuberculosis., Avishek Mitra, Ying-Hui Ko, Gino Cingolani, Michael Niederweis

Department of Biochemistry and Molecular Biology Faculty Papers

Iron is essential for growth of Mycobacterium tuberculosis (Mtb), but most iron in the human body is stored in heme within hemoglobin. Here, we demonstrate that the substrate-binding protein DppA of the inner membrane Dpp transporter is required for heme and hemoglobin utilization by Mtb. The 1.27 Å crystal structure of DppA shows a tetrapeptide bound in the protein core and a large solvent-exposed crevice for heme binding. Mutation of arginine 179 in this cleft eliminates heme binding to DppA and prevents heme utilization by Mtb. The outer membrane proteins PPE36 and PPE62 are also required for heme and hemoglobin …

Structural Basis For The Homotypic Fusion Of Chlamydial Inclusions By The Snare-Like Protein Inca., Gino Cingolani, Michael Mccauley, Anna Lobley, Alexander J Bryer, Jordan Wesolowski, Deanna L Greco, Ravi K Lokareddy, Erik Ronzone, Juan R Perilla, Fabienne Paumet

Department of Biochemistry and Molecular Biology Faculty Papers

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Here, we present the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during …

A Unique Role For Clathrin Light Chain A In Cell Spreading And Migration., Oxana M. Tsygankova, James H. Keen

Department of Biochemistry and Molecular Biology Faculty Papers

Clathrin heavy chain is the structural component of the clathrin triskelion, but unique functions for the two distinct and highly conserved clathrin light chains (CLCa and CLCb, also known as CLTA and CLTB, respectively) have been elusive. Here, we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60-80% compared to siRNA controls, and wound closure and motility by ∼50%. Surface levels of β1-integrins were unaffected by CLCa depletion. However, …

Yeast Mitochondrial Protein Pet111p Binds Directly To Two Distinct Targets In Cox2 Mrna, Suggesting A Mechanism Of Translational Activation, Julia L Jones, Katharina B. Hofmann, Andrew T. Cowan, Dmitry Temiakov, Patrick Cramer, Michael Anikin

Department of Biochemistry and Molecular Biology Faculty Papers

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in …

Trna Methylation Is A Global Determinant Of Bacterial Multi-Drug Resistance., Isao Masuda, Ryuma Matsubara, Thomas Christian, Enrique R. Rojas, Srujana S. Yadavalli, Lisheng Zhang, Mark Goulian, Leonard J. Foster, Kerwyn Casey Huang, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that m ¹ G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m ¹ G37 levels in Escherichia coli and Salmonella impair …

Gasdermin Pores Permeabilize Mitochondria To Augment Caspase-3 Activation During Apoptosis And Inflammasome Activation., Corey Rogers, Dan A. Erkes, Alexandria Nardone, Andrew E. Aplin, Teresa Fernandes-Alnemri, Emad S. Alnemri

Department of Biochemistry and Molecular Biology Faculty Papers

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME …

Loss-Of-Function Mutations In Lysyl-Trna Synthetase Cause Various Leukoencephalopathy Phenotypes, Chong Sun, Jie Song, Yanjun Jiang, Chongbo Zhao, Jiahong Lu, Yuxin Li, Yin Wang, Mingshi Gao, Jianying Xi, Sushan Luo, Meixia Li, Kevin Donaldson, Stephanie N. Oprescu, Thomas P. Slavin, Sansan Lee, Pilar L. Magoulas, Andrea M. Lewis, Lisa Emrick, Seema R. Lalani, Zhiyv Niu, Megan L. Landsverk, Magdalena Walkiewicz, Richard E. Person, Hui Mei, Jill A. Rosenfeld, Yaping Yang, Anthony Antonellis, Ya-Ming Hou, Jie Lin, Victor W. Zhang

Department of Biochemistry and Molecular Biology Faculty Papers

Objective: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene–related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.

Methods: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays.

Results: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main …

Interaction Between The Bag1s Isoform And Hsp70 Mediates The Stability Of Anti-Apoptotic Proteins And The Survival Of Osteosarcoma Cells Expressing Oncogenic Myc., Victoria J. Gennaro, Helen Wedegaertner, Steven B. Mcmahon

Department of Biochemistry and Molecular Biology Faculty Papers

BACKGROUND: The oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined.

METHODS: In our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival …

Impaired Nuclear Export Of Polyglutamine-Expanded Androgen Receptor In Spinal And Bulbar Muscular Atrophy., Frederick J. Arnold, Anna Pluciennik, Diane E. Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Prior studies have highlighted the importance of AR nuclear localization in SBMA pathogenesis; therefore, in this study, we sought to determine the role of AR nuclear export in the pathological manifestations of SBMA. We demonstrate here that the nuclear export of polyQ-expanded AR is impaired, even prior to the formation of intranuclear inclusions of aggregated AR. Additionally, we find that promoting AR export with an exogenous nuclear export signal substantially reduces its aggregation and blocks hormone-induced toxicity. Moreover, we show …

Codon-Specific Translation By M1g37 Methylation Of Trna, Ya-Ming Hou, Isao Masuda, Howard Gamper

Department of Biochemistry and Molecular Biology Faculty Papers

Although the genetic code is degenerate, synonymous codons for the same amino acid are not translated equally. Codon-specific translation is important for controlling gene expression and determining the proteome of a cell. At the molecular level, codon-specific translation is regulated by post-transcriptional epigenetic modifications of tRNA primarily at the wobble position 34 and at position 37 on the 3′-side of the anticodon. Modifications at these positions determine the quality of codon-anticodon pairing and the speed of translation on the ribosome. Different modifications operate in distinct mechanisms of codon-specific translation, generating a diversity of regulation that is previously unanticipated. Here we …

In Vitro Growth Of Human Ovarian Follicles For Fertility Preservation, Diego Marin, Min Yang, Tianren Wang

Department of Biochemistry and Molecular Biology Faculty Papers

Young female cancer survival rates significantly increased due to the great progress of cancer therapy. In fact, cryostorage and transplantation of ovarian tissue have already resulted in the birth of healthy babies. Follicle in vitro growth (IVG) has the great potential of restoring fertility by achieving functional oocytes from the most immature stages to maturation. This is suitable for a wide range of patients, from pubertal to perimenopause women. Notable achievements have been achieved in human follicle IVG in the past decade. Mature oocytes have been successfully collected from long-term sequential follicle IVG. However, it is still a major challenge …

Trypanosoma Brucei Prmt1 Is A Nucleic Acid Binding Protein With A Role In Energy Metabolism And The Starvation Stress Response., Lucie Kafková, Chengjian Tu, Kyle L. Pazzo, Kyle P. Smith, Erik W. Debler, Kimberly S. Paul, Jun Qu, Laurie K. Read

Department of Biochemistry and Molecular Biology Faculty Papers

In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed …

Highly Efficient 5' Capping Of Mitochondrial Rna With Nad+ And Nadh By Yeast And Human Mitochondrial Rna Polymerase, Jeremy G Bird, Urmimala Basu, David Kuster, Aparna Ramachandran, Ewa Grudzien-Nogalska, Atif Towheed, Douglas C. Wallace, Megerditch Kiledjian, Dmitry Temiakov, Smita S. Patel, Richard H. Ebright, Bryce E. Nickels

Department of Biochemistry and Molecular Biology Faculty Papers

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter …

Dna Methylation-Based Age Prediction And Telomere Length In White Blood Cells And Cumulus Cells Of Infertile Women With Normal Or Poor Response To Ovarian Stimulation., Scott J. Morin, Xin Tao, Diego Marin, Yiping Zhan, Jessica Landis, Jenna Bedard, Richard T. Scott, Emre Seli

Department of Biochemistry and Molecular Biology Faculty Papers

An algorithm assessing the methylation levels of 353 informative CpG sites in the human genome permits accurate prediction of the chronologic age of a subject. Interestingly, when there is discrepancy between the predicted age and chronologic age (age acceleration or "AgeAccel"), patients are at risk for morbidity and mortality. Identification of infertile patients at risk for accelerated reproductive senescence may permit preventative action. This study aimed to assess the accuracy of the "epigenetic clock" concept in reproductive age women undergoing fertility treatment by applying the age prediction algorithm in peripheral (white blood cells [WBCs]) and follicular somatic cells (cumulus cells …

Sumo-Mediated Regulation Of Nlrp3 Modulates Inflammasome Activity., Rachael Barry, Sidonie Wicky John, Gianmaria Liccardi, Tencho Tenev, Isabel Jaco, Chih-Hong Chen, Justin Choi, Paulina Kasperkiewicz, Teresa Fernandes-Alnemri, Emad S Alnemri, Marcin Drag, Yuan Chen, Pascal Meier

Department of Biochemistry and Molecular Biology Faculty Papers

The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1β release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, …

Trna 3'-Amino-Tailing For Stable Amino Acid Attachment., Howard Gamper, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others …

Control Of Ccnd1 Ubiquitylation By The Catalytic Saga Subunit Usp22 Is Essential For Cell Cycle Progression Through G1 In Cancer Cells., Victoria J. Gennaro, Timothy J. Stanek, Amy R. Peck, Yunguang Sun, Feng Wang, Shuo Qie, Karen E. Knudsen, Hallgeir Rui, Tauseef Butt, J. Alan Diehl, Steven B. Mcmahon

Department of Biochemistry and Molecular Biology Faculty Papers

Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the …

Pepducin-Mediated Cardioprotection Via Β-Arrestin-Biased Β2-Adrenergic Receptor-Specific Signaling, Laurel A. Grisanti, Toby P. Thomas, Rhonda L. Carter, Claudio De Lucia, Erhe Gao, Walter J. Koch, Jeffrey L. Benovic, Douglas G. Tilley

Department of Biochemistry and Molecular Biology Faculty Papers

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in …

Structural Basis Of Mitochondrial Transcription., Hauke S. Hillen, Dmitry Temiakov, Patrick Cramer

Department of Biochemistry and Molecular Biology Faculty Papers

The mitochondrial genome is transcribed by a single-subunit DNA-dependent RNA polymerase (mtRNAP) and its auxiliary factors. Structural studies have elucidated how mtRNAP cooperates with its dedicated transcription factors to direct RNA synthesis: initiation factors TFAM and TFB2M assist in promoter-DNA binding and opening by mtRNAP while the elongation factor TEFM increases polymerase processivity to the levels required for synthesis of long polycistronic mtRNA transcripts. Here, we review the emerging body of structural and functional studies of human mitochondrial transcription, provide a molecular movie that can be used for teaching purposes and discuss the open questions to guide future directions of …

Coupling Of Smoothened To Inhibitory G Proteins Reduces Voltage-Gated K, Lan Cheng, Moza Al-Owais, Manuel Covarrubias, Walter J. Koch, David R. Manning, Chris Peers, Natalia A Riobo-Del Galdo

Department of Biochemistry and Molecular Biology Faculty Papers

SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric G_i proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic G_iCT/TTA mice, in which G_i signaling is impaired, suggesting that the effect of SMO is mediated by G_i proteins. Investigation of the mechanism underlying the change …

Triad3a Induces The Degradation Of Early Necrosome To Limit Ripk1-Dependent Cytokine Production And Necroptosis., Norah A. Alturki, Scott Mccomb, Ardeshir Ariana, Dikchha Rijal, Robert G. Korneluk, Shao-Cong Sun, Emad S Alnemri, Subash Sad

Department of Biochemistry and Molecular Biology Faculty Papers

Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, …

The Mitochondrial Protease Htra2 Restricts The Nlrp3 And Aim2 Inflammasomes., Ian Gaël Rodrigue-Gervais, Karine Doiron, Claudia Champagne, Lindsey Mayes, Gabriel André Leiva-Torres, Paulin Vanié, Todd Douglas, Silvia M. Vidal, Emad S. Alnemri, Maya Saleh

Department of Biochemistry and Molecular Biology Faculty Papers

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson's disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude …

Breaking Symmetry In Viral Icosahedral Capsids As Seen Through The Lenses Of X-Ray Crystallography And Cryo-Electron Microscopy., Kristin N. Parent, Jason R. Schrad, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell's icosahedral symmetry, providing a gateway through the capsid. In this review, …

The Meiosis-Specific Cdc20 Family-Member Ama1 Promotes Binding Of The Ssp2 Activator To The Smk1 Map Kinase., Gregory Omerza, Chong Wai Tio, Timothy Philips, Aviva Diamond, Aaron M. Neiman, Edward Winter

Department of Biochemistry and Molecular Biology Faculty Papers

Smk1 is a meiosis-specific MAP kinase (MAPK) in budding yeast that is required for spore formation. It is localized to prospore membranes (PSMs), the structures that engulf haploid cells during meiosis II (MII). Similar to canonically activated MAPKs, Smk1 is controlled by phosphorylation of its activation-loop threonine (T) and tyrosine (Y). However, activation loop phosphorylation occurs via a noncanonical two-step mechanism in which 1) the cyclin-dependent kinase activating kinase Cak1 phosphorylaytes T207 during MI, and 2) Smk1 autophosphorylates Y209 as MII draws to a close. Autophosphorylation of Y209 and catalytic activity for substrates require Ssp2, a meiosis-specific protein that is …

Three-Dimensional Context Rather Than Nls Amino Acid Sequence Determines Importin Α Subtype Specificity For Rcc1., Rajeshwer S. Sankhala, Ravi K. Lokareddy, Salma Begum, Ruth A. Pumroy, Richard E. Gillilan, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3. This pathway is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Here we identify the mechanisms of importin α3 selectivity for RCC1. We find this isoform binds RCC1 with one order of magnitude higher affinity than the generic importin α1, although the two isoforms share an identical NLS-binding groove. Importin α3 uses its greater conformational flexibility to wedge the RCC1 β-propeller flanking the NLS against its lateral surface, preventing steric clashes with its Armadillo-core. Removing …

Synergy Of Two Low-Affinity Nlss Determines The High Avidity Of Influenza A Virus Nucleoprotein Np For Human Importin Α Isoforms., Wei Wu, Rajeshwer S. Sankhala, Tyler J Florio, Lixin Zhou, Nhan L.T. Nguyen, Ravi K. Lokareddy, Gino Cingolani, Nelly Panté

Department of Biochemistry and Molecular Biology Faculty Papers

The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates …