Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines.

UNLABELLED: Tumor necrosis factor alpha (TNF) plays a critical role in the control of Mycobacterium tuberculosis, in part by augmenting T cell responses through promoting macrophage phagolysosomal fusion (thereby optimizing CD4(+) T cell immunity by enhancing antigen presentation) and apoptosis (a process that can lead to cross-priming of CD8(+) T cells). M. tuberculosis can evade antituberculosis (anti-TB) immunity by inhibiting host cell TNF production via expression of specific mycobacterial components. We hypothesized that M. tuberculosis mutants with an increased capacity to induce host cell TNF production (TNF-enhancing mutants) and thus with enhanced immunogenicity can be useful for vaccine development. To identify mycobacterial genes that regulate host cell TNF production, we used a TNF reporter macrophage clone to screen an H37Rv M. tuberculosis cosmid library constructed in M. smegmatis The screen has identified a set of TNF-downregulating mycobacterial genes that, when deleted in H37Rv, generate TNF-enhancing mutants. Analysis of mutants disrupted for a subset of TNF-downregulating genes, annotated to code for triacylglycerol synthases and fatty acyl-coenzyme A (acyl-CoA) synthetase, enzymes that concern lipid biosynthesis and metabolism, has revealed that these strains can promote macrophage phagolysosomal fusion and apoptosis better than wild-type (WT) bacilli. Immunization of mice with the TNF-enhancing M. tuberculosis mutants elicits CD4(+) and CD8(+) T cell responses that are superior to those engendered by WT H37Rv. The results suggest that TNF-upregulating M. tuberculosis genes can be targeted to enhance the immunogenicity of mycobacterial strains that can serve as the substrates for the development of novel anti-TB vaccines. IMPORTANCE: One way to control tuberculosis (TB), which remains a major global public health burden, is by immunization with an effective vaccine. The efficacy of Mycobacterium bovis BCG, the only currently approved TB vaccine, is inconsistent. Tumor necrosis factor alpha (TNF) is a cytokine that plays an important role in controlling TB. M. tuberculosis, the causative agent of TB, can counter this TNF-based defense by decreasing host cell TNF production. This study identified M. tuberculosis genes that can mediate inhibition of TNF production by macrophage (an immune cell critical to the control of TB). We have knocked out a number of these genes to generate M. tuberculosis mutants that can enhance macrophage TNF production. Immunization with these mutants in mice triggered a T cell response stronger than that elicited by the parental bacillus. Since T cell immunity is pivotal in controlling M. tuberculosis, the TNF-enhancing mutants can be used to develop novel TB vaccines.

Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367.

Artificial transcription factors are powerful tools for regulating gene expression. Here we report results with engineered zinc-finger transcription factors (ZF-TFs) targeting four protein-coding genes, OCT4, SOX2, KLF4 and c-MYC, and one noncoding ribonucleic acid (RNA) gene, the microRNA (miRNA) miR302/367 cluster. We designed over 300 ZF-TFs whose targets lie within 1 kb of the transcriptional start sites (TSSs), screened them for increased messenger RNA or miRNA levels in transfected cells, and identified potent ZF-TF activators for each gene. Furthermore, we demonstrate that selected ZF-TFs function with alternative activation domains and in multiple cell lines. For OCT4, we expanded the target range to -2.5 kb and +500 bp relative to the TSS and identified additional active ZF-TFs, including three highly active ZF-TFs targeting distal enhancer, proximal enhancer and downstream from the proximal promoter. Chromatin immunoprecipitation (FLAG-ChIP) results indicate that several inactive ZF-TFs targeting within the same regulatory region bind as well as the most active ZF-TFs, suggesting that efficient binding within one of these regulatory regions may be necessary but not sufficient for activation. These results further our understanding of ZF-TF design principles and corroborate the use of ZF-TFs targeting enhancers and downstream from the TSS for transcriptional activation.

Downregulation of integrins by von Hippel-Lindau (VHL) tumor suppressor protein is independent of VHL-directed hypoxia-inducible factor alpha degradation.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in the majority of clear-cell renal cell carcinomas (RCCs). It was previously shown that VHL decreased the abundance of integrins alpha2, alpha5, and beta1, which is consistent with VHL-associated changes in cell-cell and cell - extracellular matrix adhesions. We investigated the mechanism by which VHL downregulates integrins. Although VHL can target hypoxia-inducible factor alpha (HIFalpha) subunits for degradation, VHL-dependent reduction of integrins was independent of O2 concentration and HIFalpha levels. VHL reduced the half-lives of integrins, and this activity was blocked by proteasomal inhibition. Although ectopically expressed FLAG-VHL retained HIFalpha degradation activity, it neither downregulated integrins nor promoted adherens and tight intercellular junctions, in contrast to expressed wild-type VHL. Moreover, integrins co-immunoprecipitated with wild-type VHL, but not FLAG-VHL. These data indicate that the downregulation of integrins by VHL is distinct from the regulation of HIFalpha subunits by VHL, and suggests that the loss of this activity contributes to VHL-associated RCC development through disruption of adherens and tight junctions.

The cysteine-rich region of T1R3 determines responses to intensely sweet proteins.

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.

[Functional di-domain of neuronal growth inhibitory factor].

Neuronal growth inhibitory factor (GIF), known also as metallothionein-III (MT-III), was the first validated to be capable of inhibiting growth of neuronal cells in nervous system, its beta-domain being functional. GIF functional di-domain (GIFbeta- beta) was constructed to study the structure and function of GIF. N terminal beta-domain and C terminal beta-domain cDNAs were amplified by PCR, inserted into vector pGEX-4T-1 and expressed in Escherichia coli, as carboxyl terminal extension of glutathione-S-transferase (GST), by IPTG induction. After digestion by thrombin, the fusion protein was isolated by passing through a glutathione-Sepharose 4B affinity chromatography column and was purified by gel fit ration on Sephacryl-S100. About 60 mg protein per liter of bacterial cell culture was achieved. The results of SDS-PAGE, amino acid composition, molecular mass, the ratio of metal/protein and sulfhydryl group/protein showed that the purified protein was the GIFbeta- beta. Circular dichroism (CD) spectroscopy show GIFbeta- beta has characteristic metal-sulfhydryl clusters of metallothionein family. Inhibitory activities detected by the MTT reduction assay are: GIF > GIFbeta-beta > GIF beta-domain.