Browsing by Subject "Amino acid sequence"
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Item Canonical and variant histones of protozoan parasites(IMR Press, 2011-06-01) Dalmasso, Maria Carolina; Sullivan, William Joseph, Jr.; Angel, Sergio Oscar; Pharmacology and Toxicology, School of MedicineProtozoan parasites have tremendously diverse lifestyles that require adaptation to a remarkable assortment of different environmental conditions. In order to complete their life cycles, protozoan parasites rely on fine-tuning gene expression. In general, protozoa use novel regulatory elements, transcription factors, and epigenetic mechanisms to regulate their transcriptomes. One of the most surprising findings includes the nature of their histones--these primitive eukaryotes lack some histones yet harbor novel histone variants of unknown function. In this review, we describe the histone components of different protozoan parasites based on literature and database searching. We summarize the key discoveries regarding histones and histone variants and their impact on chromatin regulation in protozoan parasites. In addition, we list histone genes IDs, sequences, and genomic localization of several protozoan parasites and Microsporidia histones, obtained from a thorough search of genome databases. We then compare these findings with those observed in higher eukaryotes, allowing us to highlight some novel aspects of epigenetic regulation in protists and to propose questions to be addressed in the upcoming years.Item Characterization of intrinsically disordered regions in proteins informed by human genetic diversity(PLOS, 2022-03-11) Ahmed, Shehab S.; Rifat, Zaara T.; Lohia, Ruchi; Campbell, Arthur J.; Dunker, A. Keith; Rahman, M. Sohel; Iqbal, Sumaiya; Biochemistry and Molecular Biology, School of MedicineAll proteomes contain both proteins and polypeptide segments that don't form a defined three-dimensional structure yet are biologically active-called intrinsically disordered proteins and regions (IDPs and IDRs). Most of these IDPs/IDRs lack useful functional annotation limiting our understanding of their importance for organism fitness. Here we characterized IDRs using protein sequence annotations of functional sites and regions available in the UniProt knowledgebase ("UniProt features": active site, ligand-binding pocket, regions mediating protein-protein interactions, etc.). By measuring the statistical enrichment of twenty-five UniProt features in 981 IDRs of 561 human proteins, we identified eight features that are commonly located in IDRs. We then collected the genetic variant data from the general population and patient-based databases and evaluated the prevalence of population and pathogenic variations in IDPs/IDRs. We observed that some IDRs tolerate 2 to 12-times more single amino acid-substituting missense mutations than synonymous changes in the general population. However, we also found that 37% of all germline pathogenic mutations are located in disordered regions of 96 proteins. Based on the observed-to-expected frequency of mutations, we categorized 34 IDRs in 20 proteins (DDX3X, KIT, RB1, etc.) as intolerant to mutation. Finally, using statistical analysis and a machine learning approach, we demonstrate that mutation-intolerant IDRs carry a distinct signature of functional features. Our study presents a novel approach to assign functional importance to IDRs by leveraging the wealth of available genetic data, which will aid in a deeper understating of the role of IDRs in biological processes and disease mechanisms.Item Cryo-EM structures of tau filaments from Alzheimer's disease(Springer Nature, 2017-07-13) Fitzpatrick, Anthony W.P.; Falcon, Benjamin; He, Shaoda; Murzin, Alexey G.; Murshudov, Garib; Garringer, Holly J.; Crowther, R. Anthony; Ghetti, Bernardino; Goedert, Michel; Scheres, Sjors H.W.; Pathology and Laboratory Medicine, School of MedicineAlzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease. Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.Item MED27 Variants Cause Developmental Delay, Dystonia, and Cerebellar Hypoplasia(Wiley, 2021) Meng, Linyan; Isohanni, Pirjo; Shao, Yunru; Graham, Brett H.; Hickey, Scott E.; Brooks, Stephanie; Suomalainen, Anu; Joset, Pascal; Steindl, Katharina; Rauch, Anita; Hackenberg, Annette; High, Frances A.; Armstrong-Javors, Amy; Mencacci, Niccolò E.; Gonzàlez-Latapi, Paulina; Kamel, Walaa A.; Al-Hashel, Jasem Y.; Bustos, Bernabé I.; Hernandez, Alejandro V.; Krainc, Dimitri; Lubbe, Steven J.; Van Esch, Hilde; De Luca, Chiara; Ballon, Katleen; Ravelli, Claudia; Burglen, Lydie; Qebibo, Leila; Calame, Daniel G.; Mitani, Tadahiro; Marafi, Dana; Pehlivan, Davut; Saadi, Nebal W.; Sahin, Yavuz; Maroofian, Reza; Efthymiou, Stephanie; Houlden, Henry; Maqbool, Shazia; Rahman, Fatima; Gu, Shen; Posey, Jennifer E.; Lupski, James R.; Hunter, Jill V.; Wangler, Michael F.; Carroll, Christopher J.; Yang, Yaping; Medical and Molecular Genetics, School of MedicineThe Mediator multiprotein complex functions as a regulator of RNA polymerase II-catalyzed gene transcription. In this study, exome sequencing detected biallelic putative disease-causing variants in MED27, encoding Mediator complex subunit 27, in 16 patients from 11 families with a novel neurodevelopmental syndrome. Patient phenotypes are highly homogeneous, including global developmental delay, intellectual disability, axial hypotonia with distal spasticity, dystonic movements, and cerebellar hypoplasia. Seizures and cataracts were noted in severely affected individuals. Identification of multiple patients with biallelic MED27 variants supports the critical role of MED27 in normal human neural development, particularly for the cerebellum.Item On the roles of intrinsically disordered proteins and regions in cell communication and signaling(BMC, 2021-08-30) Bondos, Sarah E.; Dunker, A. Keith; Uversky, Vladimir N.; Biochemistry and Molecular Biology, School of MedicineFor proteins, the sequence → structure → function paradigm applies primarily to enzymes, transmembrane proteins, and signaling domains. This paradigm is not universal, but rather, in addition to structured proteins, intrinsically disordered proteins and regions (IDPs and IDRs) also carry out crucial biological functions. For these proteins, the sequence → IDP/IDR ensemble → function paradigm applies primarily to signaling and regulatory proteins and regions. Often, in order to carry out function, IDPs or IDRs cooperatively interact, either intra- or inter-molecularly, with structured proteins or other IDPs or intermolecularly with nucleic acids. In this IDP/IDR thematic collection published in Cell Communication and Signaling, thirteen articles are presented that describe IDP/IDR signaling molecules from a variety of organisms from humans to fruit flies and tardigrades (“water bears”) and that describe how these proteins and regions contribute to the function and regulation of cell signaling. Collectively, these papers exhibit the diverse roles of disorder in responding to a wide range of signals as to orchestrate an array of organismal processes. They also show that disorder contributes to signaling in a broad spectrum of species, ranging from micro-organisms to plants and animals.Item Parasite-specific eIF2 (eukaryotic initiation factor-2) kinase required for stress-induced translation control(Portland Press, 2004) Sullivan, William J., Jr.; Narasimhan, Jana; Bhatti, Micah M.; Wek, Ronald C.; Pharmacology and Toxicology, School of MedicineThe ubiquitous intracellular parasite Toxoplasma gondii (phylum Apicomplexa) differentiates into an encysted form (bradyzoite) that can repeatedly re-emerge as a life-threatening acute infection (tachyzoite) upon impairment of immunity. Since the switch from tachyzoite to bradyzoite is a stress-induced response, we sought to identify components related to the phosphorylation of the alpha subunit of eIF2 (eukaryotic initiation factor-2), a well-characterized event associated with stress remediation in other eukaryotic systems. In addition to characterizing Toxoplasma eIF2alpha (TgIF2alpha), we have discovered a novel eIF2 protein kinase, designated TgIF2K-A (Toxoplasma gondii initiation factor-2kinase). Although the catalytic domain of TgIF2K-A contains sequence and structural features that are conserved among members of the eIF2 kinase family, TgIF2K-A has an extended N-terminal region that is highly divergent from other eIF2 kinases. TgIF2K-A specifically phosphorylates the regulatory serine residue of yeast eIF2alpha in vitro and in vivo, and can modulate translation when expressed in the yeast model system. We also demonstrate that TgIF2K-A phosphorylates the analogous regulatory serine residue of recombinant TgIF2alpha in vitro. Finally, we demonstrate that TgIF2alpha phosphorylation in tachyzoites is enhanced in response to heat shock or alkaline stress, conditions known to induce parasite differentiation in vitro. Collectively, this study suggests that eIF2 kinase-mediated stress responses are conserved in Apicomplexa, and a novel family member exists that may control parasite-specific events, including the clinically relevant conversion into bradyzoite cysts.Item Per Aspera ad Chaos: Vladimir Uversky’s Odyssey through the Strange World of Intrinsically Disordered Proteins(MDPI, 2023-06-19) Kulkarni, Prakash; Brocca, Stefania; Dunker, A. Keith; Longhi, Sonia; Biochemistry and Molecular Biology, School of MedicineItem The Histone Code of Toxoplasma gondii Comprises Conserved and Unique Posttranslational Modifications(American Society for Microbiology, 2013-12-10) Nardelli, Sheila C.; Che, Fa-Yun; Silmon de Monerri, Natalie C.; Xiao, Hui; Nieves, Edward; Madrid-Aliste, Carlos; Angel, Sergio O.; Sullivan, William J., Jr.; Angeletti, Ruth H.; Kim, Kami; Weiss, Louis M.; Pharmacology and Toxicology, School of MedicineEpigenetic gene regulation has emerged as a major mechanism for gene regulation in all eukaryotes. Histones are small, basic proteins that constitute the major protein component of chromatin, and posttranslational modifications (PTM) of histones are essential for epigenetic gene regulation. The different combinations of histone PTM form the histone code for an organism, marking functional units of chromatin that recruit macromolecular complexes that govern chromatin structure and regulate gene expression. To characterize the repertoire of Toxoplasma gondii histone PTM, we enriched histones using standard acid extraction protocols and analyzed them with several complementary middle-down and bottom-up proteomic approaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and/or electron transfer dissociation (ETD) fragmentation. We identified 249 peptides with unique combinations of PTM that comprise the T. gondii histone code. T. gondii histones share a high degree of sequence conservation with human histones, and many modifications are conserved between these species. In addition, T. gondii histones have unique modifications not previously identified in other species. Finally, T. gondii histones are modified by succinylation, propionylation, and formylation, recently described histone PTM that have not previously been identified in parasitic protozoa. The characterization of the T. gondii histone code will facilitate in-depth analysis of how epigenetic regulation affects gene expression in pathogenic apicomplexan parasites and identify a new model system for elucidating the biological functions of novel histone PTM. Importance: Toxoplasma gondii is among the most common parasitic infections in humans. The transition between the different stages of the T. gondii life cycle are essential for parasite virulence and survival. These differentiation events are accompanied by significant changes in gene expression, and the control mechanisms for these transitions have not been elucidated. Important mechanisms that are involved in the control of gene expression are the epigenetic modifications that have been identified in several eukaryotes. T. gondii has a full complement of histone-modifying enzymes, histones, and variants. In this paper, we identify over a hundred PTM and a full repertoire of PTM combinations for T. gondii histones, providing the first large-scale characterization of the T. gondii histone code and an essential initial step for understanding how epigenetic modifications affect gene expression and other processes in this organism.Item The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits(Elsevier, 2005) Rider, S. Dean, Jr.; Cai, Xiaomin; Sullivan, William J., Jr.; Smith, Aaron T.; Radke, Jay; White, Michael; Zhu, Guan; Pharmacology and Toxicology, School of MedicineVery little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.