Protein chaperones mediating copper insertion into the CuA site of the aa3-type cytochrome c oxidase of Paracoccus denitrificans.

The biogenesis of the mitochondrial cytochrome c oxidase is a complex process involving the stepwise assembly of its multiple subunits encoded by two genetic systems. Moreover, several chaperones are required to recruit and insert the redox-active metal centers into subunits I and II, two a-type hemes and a total of three copper ions, two of which form the CuA center located in a hydrophilic domain of subunit II. The copper-binding Sco protein(s) have been implicated with the metallation of this site in various model organisms. Here we analyze the role of the two Sco homologues termed ScoA and ScoB, along with two other copper chaperones, on the biogenesis of the cytochrome c oxidase in the bacterium Paracoccus denitrificans by deleting each of the four genes individually or pairwise, followed by assessing the functionality of the assembled oxidase both in intact membranes and in the purified enzyme complex. Copper starvation leads to a drastic decrease of oxidase activity in membranes from strains involving the scoB deletion. This loss is shown to be of dual origin, (i) a severe drop in steady-state oxidase levels in membranes, and (ii) a diminished enzymatic activity of the remaining oxidase complex, traced back to a lower copper content, specifically in the CuA site of the enzyme. Neither of the other proteins addressed here, ScoA or the two PCu proteins, exhibit a direct effect on the metallation of the CuA site in P. denitrificans, but are discussed as potential interaction partners of ScoB.

Leukocyte ß7 integrin targeted by Krüppel-like factors.

Constitutive expression of Krüppel-like factor 3 (KLF3, BKLF) increases marginal zone (MZ) B cell numbers, a phenotype shared with mice lacking KLF2. Ablation of KLF3, known to interact with serum response factor (SRF), or SRF itself, results in fewer MZ B cells. It is unknown how these functional equivalences result. In this study, it is shown that KLF3 acts as transcriptional repressor for the leukocyte-specific integrin ß7 (Itgb7, Ly69) by binding to the ß7 promoter, as revealed by chromatin immunoprecipitation. KLF2 overexpression antagonizes this repression and also binds the ß7 promoter, indicating that these factors may compete for target sequence(s). Whereas ß7 is identified as direct KLF target, its repression by KLF3 is not connected to the MZ B cell increase because ß7-deficient mice have a normal complement of these and the KLF3-driven increase still occurs when ß7 is deleted. Despite this, KLF3 overexpression abolishes lymphocyte homing to Peyer's patches, much like ß7 deficiency does. Furthermore, KLF3 expression alone overcomes the MZ B cell deficiency when SRF is absent. SRF is also dispensable for the KLF3-mediated repression of ß7. Thus, despite the shared phenotype of KLF3 and SRF-deficient mice, cooperation of these factors appears neither relevant for the formation of MZ B cells nor for the regulation of ß7. Finally, a potent negative regulatory feedback loop limiting KLF3 expression is shown in this study, mediated by KLF3 directly repressing its own gene promoter. In summary, KLFs use regulatory circuits to steer lymphocyte maturation and homing and directly control leukocyte integrin expression.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.