Interaction between ribosomal proteins S1 and S2

Zu Beginn des neuen Milleniums ist es gelungen die molekulare Struktur der ribosomalen Untereinheiten und des gesamten Ribosoms von Escherichia coli aufzuklären. Im Gegensatz dazu konnte die Struktur des essentiellen ribo-somalen Proteins S1 und seine Bindestelle an der kleinen Untereinheit des bakteriellen Ribosomes aufgrund seiner hohen Flexibilität noch nicht bestimmt werden. Da Protein S1 für die Initiation der Translation in allen bisher bekannten Gram-negativen Bakterien notwendig ist, war das Ziel dieser Studie die strukturelle Charakterisierung dieses Proteines, mit einem speziellen Schwerpunkt auf die Interaktion mit dem Ribosom. In vorangegangenen Studien wurde gezeigt, dass für die Bindung von S1 an das Ribosom das Protein S2 benötigt wird. In der vorliegenden Arbeit konnte ich zeigen, dass diese Interaktion durch die N-terminale Domäne von S1 vermittelt wird, wobei der N-Terminus direkt mit der sogenannten „coiled-coil“ Domäne des ribosomalen Proteins S2 interagiert. Mithilfe von NMR-Studien konnte ich zeigen, dass die Kernstruktur der N-terminalen Domäne von S1 aus vier ß-Strängen aufgebaut ist, die von flexiblen Regionen am N- und am C-Terminus flankiert sind. Überaschenderweise deuten die Ergebnisse meiner Untersuchungen darauf hin, dass der flexible N-Terminus bestehend aus 18 Aminosäuren (hier als S118 bezeichnet) essentiell für die Bindung von nativem Protein S1 und verkürzten S1-Varianten an das Ribosom ist. Weitere Studien zeigen, dass dieses S118 Peptid an das Ribosom bindet und mit dem nativen Protein S1 um die Bindestelle kompetitiert. Zusätzlich weisen meine Ergebnisse darauf hin, dass die „coiled-coil“-Domäne des ribosomalen Proteins S2 notwendig, aber auch ausreichend für die Assemblierung von S1 an das Ribosom ist. Interessanterweise zeigen Mutationsanalysen an Protein S2, dass die Aminosäuren Asparagin an Position 145 und Glycin an Positionen 148 und 149 in der „coiled-coil“ Domäne in sterischer Nähe zur globulären Domäne von Protein S2 wichtig für diese Bindung ist, da Mutationen an diesen Stellen die Interaktion mit S1 verhindert. Zusammengefasst, konnte ich in meinen Studien die Bindung zwischen den ribosomalen Proteinen S1 und S2 näher charakterisieren und die Interaktionsdomänen eingrenzen. Die Ergebnisse meiner Untersuchungen weisen darauf hin, dass der flexible Bereich am N-Terminus von S1 eine primäre Interaktionsdomäne mit der kleinen Untereinheit des Ribosomes darstellt. Es ist denkbar, dass dieser Bereich durch seine hohe intrinsische Flexibilität über einen „induced-fit“ Mechanismus mit der Region zwischen der „coiled-coil“-Domäne und der globulären Domäne des ribosomalen Proteins S2 interagiert. Da diese Bindung essentiell für das Überleben von Gram-negativen Bakterien ist, ist es vorstellbar, dass dieser Bereich ein potentielles Angriffsziel für die Entwicklung von neuen antimikrobiellen Wirkstoffen darstellen könnten, die semi-selektiv gegen Gram-negative pathogene Bakterien wirken. Weiters könnten diese Wirkstoffe die nützliche Gram-positive Flora nicht zerstören, da diese Bakterien kein homologes Protein S1 besitzen.The structure of the E. coli ribosome is solved at atomic resolution. In con-trast, hitherto the position and structure of the essential ribosomal protein S1 has not been determined due to its intrinsic flexibility. Since protein S1 is pivotal for translation initiation in all Gram-negative bacteria studied so far, the aim of this project was the structural characterization of protein S1 with a special focus on the site of interaction with the ribosome. Previously, we have obtained evidence that protein S1 requires protein S2 for binding to the 30S ribosomal subunit. In this study, I was able to show that assembly of protein S1 to the ribosome is me-diated by its N-terminal domain D1 that directly interacts with the coiled-coil do-main of protein S2. Using an NMR-based approach, I determined that the N-terminal domain D1 consists of a folded core of four β-strands that are flanked by flexible N- and C-terminal regions. Surprisingly, the flexible N-terminal region of domain D1 of protein S1 comprising eighteen amino acids (referred to as S118) is indispensable for binding of protein S1 and its truncated variants to the ribo-some. Moreover, I showed that peptide S118 binds to the ribosome and competes with native protein S1 for its binding pocket on the 30S ribosomal subunit. In addition, my results indicate that the coiled-coil domain of protein S2 is required and sufficient to allow binding of protein S1 to the ribosome. Noteworthy, changing residue Asn145 of protein S2, the side chain of which is oriented towards the cleft of the head, body, and neck of the 30S ribosomal subunit where protein S1 has been proposed to bind, abrogates the interaction between the coiled-coil domain of protein S2 and the N-terminal region of protein S1. Likewise, two glycine residues of the coiled-coil domain located close to the globular domain of S2 are required for the interaction with the N-terminus of protein S1, since glycine to alanine mutations at these positions (Gly148 and Gly149) also abolish the interaction between proteins S1 and S2. Taken together, my data support the notion that the flexible region of eigh-teen amino acids in length located at the N-terminus of protein S1 could serve as a primary interaction site for S1 on the 30S subunit. Due to its intrinsic flexibil-ity the S118 region could act as an anchoring domain, which interacts specifi-cally with residues at the boundary between the coiled-coil and globular domain of protein S2 via an induced fit mechanism. Thus, during the course of these studies I was able to narrow down the site of interaction between proteins S1 and S2. Moreover, I identified several residues which might be directly involved in this interaction. Since assembly of protein S1 to the ribosome is essential for the viability of Gram-negative bacteria, this interaction surface might serve as potential target for the design of novel antimicrobial compounds that act semi-selective against Gram-negative pathogens without affecting the Gram-positive flora, which do not harbor functional homologues of protein S1

Quantitative analysis of mutant subclones in chronic myeloid leukemia : comparison of different methodological approaches

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points

ДООПЕРАЦИОННАЯ ДИАГНОСТИКА И ХИРУРГИЧЕСКОЕ ЛЕЧЕНИЕ УЩЕМЛЕННОЙ ГРЫЖИ ВИНСЛОВА ОТВЕРСТИЯ

The case of timely diagnosis and surgical treatment of a rare strangulated hernia through the foramen of Winslow is reported.Представлен случай своевременной диагностики и успешного хирургического лечения редко встречающейся ущемленной грыжи Винслова отверстия

ХРОНИЧЕСКИЙ АППЕНДИЦИТ. ОПРЕДЕЛЕНИЕ ПОНЯТИЯ, ДИАГНОСТИКА И ЛЕЧЕНИЕ

The literature review focuses on definition  of “chronic appendicitis”, clinical manifestations of the disease, and differential  diagnosis  in the presence of chronic pain in the right iliac area. The article also considers  indications and effectiveness for instrumental diagnostic  methods (irrigoscopy, ultrasonography, multi-slice  computed tomography, laparoscopy), and the adequacy  of surgical treatment for chronic appendicitis.Обзор литературы посвящен дефиниции понятия «хронический аппендицит», клиническим проявлениям болезни, дифференциальной диагностике при хронических болях в правой подвздошной области. Обсуждаются показания  к применению  методов  инструментальной  диагностики (ирригоскопия, ультразвуковое  исследование,  мультиспиральная компьютерная томография, лапароскопия) и их эффективность; обоснованность  назначения  хирургического лечения  хронического аппендицита

Remote control of gene function by local translation

The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function

CHRONIC APPENDICITIS. DEFINITION, DIAGNOSING AND TREATMENT

The literature review focuses on definition  of “chronic appendicitis”, clinical manifestations of the disease, and differential  diagnosis  in the presence of chronic pain in the right iliac area. The article also considers  indications and effectiveness for instrumental diagnostic  methods (irrigoscopy, ultrasonography, multi-slice  computed tomography, laparoscopy), and the adequacy  of surgical treatment for chronic appendicitis