E. coli metabolic protein aldehydealcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosom

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets

A creature with a hundred waggly tails: intrinsically disordered proteins in the ribosome

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Intrinsic disorder (i.e., lack of a unique 3-D structure) is a common phenomenon, and many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions constitute a significant part of all proteomes, and their functional repertoire is complementary to functions of ordered proteins. In fact, intrinsic disorder represents an important driving force for many specific functions. An illustrative example of such disorder-centric functional class is RNA-binding proteins. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 3,411 ribosomal proteins from 32 species. We show that many ribosomal proteins are intrinsically disordered or hybrid proteins that contain ordered and disordered domains. Predicted globular domains of many ribosomal proteins contain noticeable regions of intrinsic disorder. We also show that disorder in ribosomal proteins has different characteristics compared to other proteins that interact with RNA and DNA including overall abundance, evolutionary conservation, and involvement in protein–protein interactions. Furthermore, intrinsic disorder is not only abundant in the ribosomal proteins, but we demonstrate that it is absolutely necessary for their various functions

The Characterization of the Transcription Factor Msab and Its Role in Staphylococcal Virulence

Staphylococcus aureus is a common human pathogen that is responsible for a wide range of infections, ranging from relative minor skin infections to life-threatening disease such as bacteremia, septicemia, and endocarditis. S. aureus possesses many different virulent factors that aid in its ability to cause this wide array of infections. One major virulence factor includes the production of capsular polysaccharide (CP). The production of CP plays a major role in the virulence response during infection specifically by providing S. aureus an antiphagocytic mechanism that allows the pathogen to evade phagocytosis during an infection. S. aureus has developed complex genetic regulatory systems responsible for controlling virulence factors including CP. The production of CP is encoded by a 16-gene operon, which is regulated in response to several environmental stimuli. In detail, CP is produced in the late- and post-exponential growth phases in vitro, but not in the early- or mid-exponential growth phases. Several genetic regulators have been identified to be involved in controlling CP production, but the mechanisms involved in this regulation of cap are still poorly understood. In this work, we describe that the msaABCR operon significantly regulates CP production. We found that a protein product of the msaABCR transcript, MsaB, directly binds to a nucleotide repeat upstream of the promoter region of the cap operon, termed MsaB binding site or (MBS), as a transcriptional activator of CP production. Furthermore, we show that even though MsaB is expressed throughout all four exponential growth phases, it only activates CP production in the late- and post-exponential growth phases. Several other regulators have also been shown to directly bind within the cap promoter region. We examined potential interactions between MsaB and two other nutrient-sensing regulators, CodY and CcpE, binding the cap promoter. From the findings of this work, we have concluded that MsaB (activator) and CodY (repressor) have nutrient-dependent competitive interactions binding to the cap promoter. Findings from this work has led us to explore targeting and inhibiting MsaB transcriptional regulation as a potential novel therapeutic target in combating S. aureus

Signal transduction and Rickettsial infection of tick cells

Spotted fever group (SFG) Rickettsia are obligate intracellular bacteria that are carried by ticks. One such tick, Dermacentor variabilis is a vector for the etiologic agent of Rocky Mountain spotted fever, R. rickettsii. These ticks also carry a non-pathogenic R. montanensis, the agent used in this study. Interestingly, field data collected from infected D. variabilis throughout the United States revealed that the majority of Rickettsia in ticks are non-pathogenic species such as R. montanensis. Although ticks serve as both vector and reservoir hosts for SFG Rickettsia, many questions regarding tick-Rickettsia interaction remain unresolved. Therefore, the overall goal of this research was to study the relationship between ticks and Rickettsia, specifically examining the molecular mechanisms of rickettsial infection of tick host. As SFG Rickettsia can move between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for invasion of both types of host cell. Biochemical inhibition assays revealed that the tick molecules, PI 3-kinase, protein tyrosine kinases, Src, FAK, Rho GTPase Rac1, N-WASP, Arp2/3 complex, actin, and V-ATPase are important for R. montanensis invasion. Further studies were executed to molecularly and functionally characterize the tick molecules, Arp2/3 complex and V-ATPase, which are central to rickettsial internalization. Full length cDNA of Arp2/3 complex subunits and V-ATPase from D. variabilis were isolated. Transcriptional profiles of Arp2/3 complex subunits and V-ATPase showed greater expression of the mRNA in tick ovaries compared to midgut and salivary glands. In addition, to gain insight into rickettsial invasion in nature, Arp2/3 complex inhibition assays were performed in tick tissues. The results demonstrated the involvement of Arp2/3 complex in rickettsial entry into midgut, ovary, and salivary glands. The tick molecules identified in this study may provide novel points of intervention in the transmission of tick-borne rickettsial diseases

Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot

Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot