ClinicalTrials.govNCT01020838.

B1 cells are an important cell population for the production of natural antibodies and for antibacterial immunoglobulin responses. Here we identified the mouse protein Siglec-G as a B1 cell inhibitory receptor. Siglec-G was expressed in a B cell-restricted way, with large amounts present in B1 cells. When overexpressed, Siglec-G inhibited B cell receptor-mediated calcium signaling. Siglec-G-deficient mice had massive expansion of the B1a cell population, which began early in development and was B cell intrinsic. Siglec-G-deficient mice had higher titers of natural IgM antibodies but not a higher penetrance of IgG autoantibodies. Siglec-G-deficient B1 cells showed a strongly enhanced calcium signaling. Our results demonstrate that Siglec-G-dependent negative regulation exists in B1 cells, which may explain the naturally muted signaling response of B1 cells.

Newly synthesized proteins often require the assistance of molecular chaperones to efficiently fold into functional three-dimensional structures. At first, ribosome-associated chaperones guide the initial folding steps and protect growing polypeptide chains from misfolding and aggregation. After that folding into the native structure may occur spontaneously or require support by additional chaperones which do not bind to the ribosome such as DnaK and GroEL. Here we review the current knowledge on the best-characterized ribosome-associated chaperone at present, the Escherichia coli Trigger Factor. We describe recent progress on structural and dynamic aspects of Trigger Factor's interactions with the ribosome and substrates and discuss how these interactions affect co-translational protein folding. In addition, we discuss the newly proposed ribosome-independent function of Trigger Factor as assembly factor of multi-subunit protein complexes. Finally, we cover the functional cooperation between Trigger Factor, DnaK and GroEL in folding of cytosolic proteins and the interplay between Trigger Factor and other ribosome-associated factors acting in enzymatic processing and translocation of nascent polypeptide chains.

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H2O2), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1-5 mM H2O2 for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H2O2 and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H2O2 in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia.

Although there is increasing evidence that blood-derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf-2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2-like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor-educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy. V V C

A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp–protease complex.

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryoelectron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.

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