Aryl Hydrocarbon Receptor Interacting Protein Maintains Germinal Center B Cells through Suppression of BCL6 Degradation

B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a cochaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma

The gp38 Adhesins of the T4 Superfamily: A Complex Modular Determinant of the Phage’s Host Specificity

The tail fiber adhesins are the primary determinants of host range in the T4-type bacteriophages. Among the indispensable virion components, the sequences of the long tail fiber genes and their associated adhesins are among the most variable. The predominant form of the adhesin in the T4-type phages is not even the version of the gene encoded by T4, the archetype of the superfamily, but rather a small unrelated protein (gp38) encoded by closely related phages such as T2 and T6. This gp38 adhesin has a modular design: its N-terminal attachment domain binds at the tip of the tail fiber, whereas the C-terminal specificity domain determines its host receptor affinity. This specificity domain has a series of four hypervariable segments (HVSs) that are separated by a set of highly conserved glycine-rich motifs (GRMs) that apparently form the domain’s conserved structural core. The role of gp38’s various components was examined by a comparative analysis of a large series of gp38 adhesins from T-even superfamily phages with differing host specificities. A deletion analysis revealed that the individual HVSs and GRMs are essential to the T6 adhesin’s function and suggests that these different components all act in synergy to mediate adsorption. The evolutionary advantages of the modular design of the adhesin involving both conserved structural elements and multiple independent and easily interchanged specificity determinants are discussed

The viral protein Rki: an atypical cochaperone partner of Hsp70

Les chaperons moléculaires de la famille Hsp70 sont des protéines ubiquitaires qui interviennent dans de nombreux processus cellulaires liés au repliement des protéines. Les fonctions d'Hsp70 sont indissociables de la présence des cochaperons JDP (J-domain protein). Ces protéines, grâce à leur J-domaine caractéristique, stimulent l'activité ATPasique de Hsp70, facilitent leur prise en charge des substrats et leur confèrent leur localisation cellulaire. L'analyse de la séquence du génome de l'entérobactriophage RB43, membre de la famille du bacteriophage T4, a révélé la présence d'un gène codant une JDP putative (l'orfan 057w). Dans ce travail, nous avons montré que le J-domaine de cette JDP, nommée Rki, est fonctionnel in vivo et que Rki interagit spécifiquement avec le chaperon multifonctionnel Hsp70/DnaK de l'hôte Escherichia coli. Cependant, à la différence des trois cochaperons JDP de DnaK présents chez E. coli, la protéine Rki ne fonctionne pas comme un cochaperon de type général de DnaK in vivo et in vitro. Au contraire, l'expression de Rki est fortement toxique dans une souche sauvage d'E. coli. De façon remarquable, cette toxicité est totalement abolie en l'absence de DnaK endogène ou quand le J-domaine de Rki est inactivé. D'autres expériences in vivo ont ensuite révélé que Rki est exprimée précocement durant l'infection par RB43 et que la délétion du gène rki diminue significativement la prolifération du bactériophage. De plus, nous avons trouvé que des mutations dans le gène dnaK de l'hôte suppriment efficacement le phénotype de retard de croissance du phage muté pour le gène rki, indiquant que Rki interfère spécifiquement avec les fonctions cellulaires de DnaK. Enfin, nous avons montré que l'interaction de Rki avec le chaperon DnaK de l'hôte stabilise rapidement le facteur s32 de réponse au choc thermique, qui est normalement adressé à la dégradation par DnaK.The highly conserved molecular chaperone Hsp70 is involved in a plethora of cellular processes associated with protein folding. To function as a molecular chaperone, Hsp70 requires the presence of its obligate J-domain cochaperone partners (JDP). These proteins, thanks to their J-domain signature, stimulate Hsp70's ATPase activity, facilitate substrate delivery and confer specific cellular localization to Hsp70. Genome analysis of the T4-like enterobacteriophage RB43, revealed a gene encoding a putative JDP (orfan 057w). In this work, we first show that the J-domain of this JDP, named Rki, is functional in vivo. Moreover, we show that Rki specifically interacts with the E. coli host multifunctional Hsp70/DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki full-length does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor s32, which is normally targeted for degradation by DnaK

La protéine virale Rki (un partenaire atypique du chaperon Hsp70)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Chaperone-assisted Excisive Recombination, a Solitary Role for DnaJ (Hsp40) Chaperone in Lysogeny Escape

International audienceTemperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not requirea functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development

YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.

International audienc

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ³² of <em>Escherichia coli</em>

<div><p>The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene <em>057w</em> of the T4-related enterobacteriophage RB43 encodes a <em>bona fide</em> JDP protein, named Rki, which specifically interacts with the <em>Escherichia coli</em> host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by <em>E. coli</em>, Rki does not act as a generic cochaperone <em>in vivo</em> or <em>in vitro</em>. Expression of Rki alone is highly toxic for wild-type <em>E. coli</em>, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further <em>in vivo</em> analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the <em>rki</em> gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host <em>dnaK</em> gene efficiently suppress the growth phenotype of the RB43 <em>rki</em> deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ³², which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ³² facilitates RB43 bacteriophage proliferation is discussed.</p> </div

Rki is transcribed early during bacteriophage infection.

<p>(A) Early promoter mapping. The WebLogo for RB43 early promoter was determined as described in Nolan <i>et al</i>., 2006. The G nucleotide underlined is the putative transcription start site. Putative up elements and the −10 and −35 region are boxed. (B) Northern blot analysis showing transcription of <i>rki</i> and two control genes known to be transcribed in the early (<i>g43</i>) or late (<i>g37.2</i>) phase of infection. (C) Western blot analysis of whole cell extracts prepared from W3110 cells non-infected (−) or infected with RB43 at a MOI ∼10, during 10, 20, 30, or 60 min at 30°C and revealed using an anti-Rki rabbit antibody.</p