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

Trattamenti e ricoprimenti superficiali per migliorare il comportamento tribologico di viti in acciaio

I collegamenti filettati, tra cui i giunti bullonati, sono ampiamente utilizzati in applicazioni meccaniche per la loro facilità di montaggio e smontaggio nel momento in cui fossero necessarie operazioni di manutenzione o riparazione. Sia il livello che la stabilità dei carichi di serraggio, che vengono generati dalla coppia di serraggio, governano la sicurezza e l’affidabilità di questi dispositivi. Gli aspetti tribologici della fase di serraggio sono fondamentali per definire il legame coppia-precarico, in quanto all’atto del serraggio una parte dell'energia viene spesa per vincere l'attrito che si manifesta nei piani di appoggio sottotesta, mentre il resto serve per vincere la resistenza d'attrito della filettatura (vite-madrevite), comprimendo le parti da collegare e mettendo quindi in tensione la vite. Inesattezze sui coefficienti d’attrito possono portare alla sovrastima o sottostima del precarico e quindi anche delle prestazioni funzionali del giunto filettato. L'obiettivo quindi è diminuire il più possibile questi coefficienti d’attrito, poiché a parità di coppia di serraggio il precarico sulla vite risulta maggiore, a vantaggio della stabilità del collegamento. A questo scopo vengono analizzate una serie di prove sperimentali (da bibliografia scientifica) su trattamenti e ricoprimenti superficiali (shot peening, DLC, bisolfuro di molibdeno, nitruro e carbonitruro di titanio, zincatura, brunitura) ponendo l'attenzione principalmente sui risultati relativi ad attrito e usura; nell'ultima parte vengono indicate delle possibili applicazioni di questi trattamenti e ricoprimenti superficiali alle viti in acciaio, al fine di migliorare il loro comportamento tribologico

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Protein S1₁₀₆ affects <i>E. coli</i> growth by displacing native S1 from the ribosome.

<p>(<b>A</b>) In contrast to synthesis of protein S1_87–194 (representing domain D2, -•-), synthesis of S1₁₀₆ and S1₁₉₄ (representing domains D1 (-▪-) and D1-2 (-▴-)) inhibits bacterial growth. <i>E. coli</i> strain JE28 harbouring plasmids pProEX-HTb (-◊-), pPro-S1D1F (-▪-), pPro-S1D2F (-•-) and pPro-S1D1-2F (-▴-) were grown in LB medium containing ampicilin (100 µg/ml) and kanamycin (20 µg/ml). At OD₆₀₀ of 0.2–0.25 (indicated by an arrow) 50 µM IPTG was added to the cultures. Aliquots were withdrawn from each culture for ribosome preparation 1 hour upon induction. (<b>B</b>) Proteins present in S30 extracts (lanes 1, 3, 5, and 7) and 70 S ribosomes (lanes 2, 4, 6, and 8) prepared from cells without overexpression (lanes 1 and 2), and cells overexpressing S1₁₀₆ (lanes 3 and 4), S1_87–194 (lanes 5 and 6), or S1₁₉₄ (Lanes 7 and 8) were separated on a 12.5% SDS-PAGE and presence of protein S1 and its variants on 70 S ribosomes was checked by western blot analysis using anti-S1 antibodies (panel a), anti-FLAG (panel b) and anti-L2 antibodies (panel c), which served as loading control. The positions of the respective proteins are indicated to the right. (<b>C</b>) The N-terminal domain of S1 is required for assembly to the ribosome. Equimolar amounts of HIS-tagged ribosomes (lanes 1 and 3) and ribosome free S100 extract (lanes 2 and 4) purified from <i>E. coli</i> strain JE28 overexpressing FLAG-tagged proteins S1 (lanes 1 and 2) and S1_87–557, lacking domain D1 (lanes 3 and 4) were separated on a 12.5% SDS-PAGE. The presence of S1 and S1_87–557 was determined by western blot analysis employing anti-FLAG antibodies (panel a) and anti-L2 antibodies (panel b), which served as loading control.</p

Synthesis of S1 variants S1₁₀₆ and S1₁₉₄ results in selective translation of lmRNAs.

<p>Pulse labelling of strain JE28 harbouring plasmids encoding proteins S1₁₀₆ (lanes 1–4), S1_87–194 (lanes 5–8), and S1₁₉₄ (lanes 9–12) was carried out before (time point 0) and 15, 30, and 60 min upon induction as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032702#s4" target="_blank">Materials and Methods</a>. Labelled proteins were separated on a 12.5% SDS-PAGE. Positions of proteins S1₁₀₆, S1_87–194 and S1₁₉₄ (marked by asterisks) and the position of the CI-LacZ fusion protein encoded by a lmRNA are indicated to the right of the autoradiograph.</p

Domain D2 is not involved in ribosome binding of protein S1.

<p>(<b>A</b>) Ribosome binding of proteins S1 or S1_Δ103–181 was determined 60 minutes upon induction of their synthesis in strain JE28 harbouring either plasmid pPro-S1F (lanes 1 and 2) or pPro-S1ΔD2F (lanes 3 and 4). S30 extracts (lanes 1 and 3) and purified 70 S ribosomes (lanes 2 and 4) were loaded on SDS-PAGE. The positions of proteins S1 and S1_Δ103–181 are indicated to the right. (<b>B</b>) The binding of S1 (lanes 5–7) or S1_Δ103–181 (lanes 8–10) for the ribosome was determined by <i>in vitro</i> reconstitution experiments employing 30 S(-S1) subunits. The affinity of both proteins was directly compared by a competition experiment incubating 30 S (-S1) ribosomes concomitantly with both proteins S1 and S1_Δ103–181 in equimolar amounts (lanes 12–14). Upon incubation the ribosomes were separated from unbound proteins as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032702#s4" target="_blank">Material and Methods</a>, and the proteins present in the different fractions were separated on SDS-PAGE and visualized by Coomassie staining. I, input (lanes 2, 5, 8, and 12); R, ribosome fractions (lanes 3, 6, 9, and 13); FT, flow through fractions (lanes 4, 7, 10, and 14). 30 S, 30 S ribosomes before depletion for protein S1 (lane 15); 30 S(-S1), S1 depleted ribosomes used for the study (lane 16). The positions of proteins S1 and S1_Δ103–181 are indicated to the right. Lanes 1 and 11, protein size marker.</p