SummaryProtein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit a-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD + -specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

UruguayMicroRNAs (miRNAs) are a novel class of small noncoding RNA molecules that regulate gene expression by inducing degradation or translational inhibition of target mRNAs. There are more than 500 miRNA genes reported in the human genome, constituting one of the largest classes of regulatory genes. Increasing experimental evidence supports the idea of aberrant miRNA expression in cancer pathogenesis. We analyzed the pattern of miRNA expression in chronic lymphocytic leukemia (CLL) cells and our results showed a global reduction in miRNA expression levels in CLL cells associated to a consistent underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are differentially expressed between patients with different clinical outcomes. Five novel miRNA candidates cloned from leukemic cells are reported. Surprisingly, predicted mRNA targets for these novel miRNA revealed a high proportion of targets located in a small region of chromosome 1, which is frequently altered in human cancer. Additionally, several targets were shared by at least two of miRNA candidates. Predicted targets included several genes recently described as tumor suppressors. These data could afford new avenues for exploring innovative pathways in CLL biology and therapy.

IntroductionAfter gene rearrangement, immunoglobulin (Ig) variable genes are diversified by somatic hypermutation (SHM), whereas the effector function of the constant domain is modified by class switch recombination (CSR). These processes depend on activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme expressed in B cells from secondary lymphoid organs on CD40 ligand (CD40L) stimulation. 1 Given that the absence of AID expression in one form of the hyper-IgM syndrome in humans 2 and in AID-targeted mice abolishes CSR and SHM, this protein is thought to play a major role in both processes. 3 Fifty percent of patients with chronic lymphocytic leukemia (CLL) display mutated V H genes. 4 The mutational profile of immunoglobulin genes represents an important prognostic factor 5,6 because patients expressing unmutated V H genes exhibit poor prognoses. In addition, previous reports 7-9 have demonstrated that CSR frequently occurs in CLL and predominates in unmutated B-cell CLLs (B-CLLs).In this work, we have examined the expression of AID transcripts, SHM, and CSR in 65 patients with CLL expressing either unmutated (33 of 65) or mutated (32 of 65) V H genes. Our results show that patients with unmutated B-CLL can constitutively express AID transcripts. This fact is associated with the presence of mutations in the preswitch DNA region and with an active CSR, but it is not related to the SHM process. Additionally, in patients with mutated CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Study design Healthy and CLL samplesBlood was collected from 4 healthy controls and 65 typical CLL patients from Hôtel-Dieu, Pitié-Salpêtrière, and Pasteur hospitals (Paris, France), Sao Paulo and Servidor Publico Estadual hospitals (Sao Paulo, Brazil), and Hospital Maciel (Montevideo, Uruguay). B cells were purified through negative depletion by using RosetteSep antibody cocktail (StemCell Technologies, Vancouver, BC, Canada) and were stimulated in vitro for 5 days with 1 g/mL recombinant soluble CD40L (Immunex, Seattle, WA) and 1000 U/mL interleukin (IL)-4 (PharMingen, San Diego, CA). Analysis of AID transcriptsPolymerase chain reaction (PCR) amplification of CLL cDNA was carried out as described previously, 2 and a semiquantitative protocol for AID expression was performed. Briefly, cDNA obtained from 5 ϫ 10 6 B cells was amplified by 20 cycles of PCR using AID 2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) 10 primers. The fragments obtained were transferred and hybridized with specific AID and GAPDH ␣-[ 32 P] dCTP (deoxycytidine-5Ј-triphosphate)-labeled probes. Analysis of CSR processPCR amplification of different isotype transcripts was performed as described by Oppezzo et al. 9 Circle isotype-specific (I-C) transcripts, termed circle transcripts (CTs), were analyzed by PCR with primers I-␥ (forward) and C-(reverse), as described by Kinoshita et al. 11 Mutation analysis of V H and preswitch regions Sequences of V H genes were det...

Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V H and V L sequences, but expressing different isotypes (IgA1 PER and IgG1 PER ), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C H 1 domain in structuring the Ag-binding site into a more kinetically competent form. ( J. Clin. Invest. 1996. 98:2235-2243.)

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Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process.

Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcgR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigenspecific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors-associated g chain (FcR-g)-deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-g chainexpressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcgR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells. Cancer Res; 71(15); 5134-43. Ó2011 AACR.