Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

Regulation of cancer aggressive features in melanoma cells by microRNAs

MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer. A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells. Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells. To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities. This screening revealed two major cohorts of differentially expressed miRNAs. We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive. This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma. Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice. Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings. Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.     

Ca-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca^2 + across the OMM and because Ca^2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca^2 + levels ([Ca^2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca^2 + and induce VDAC1 over-expression. Direct elevation of [Ca^2 +]i by the Ca^2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca^2 +]i using the cell-permeable Ca^2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca^2 +]i levels and apoptosis induction, as induced by H₂O₂ or As₂O₃, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca^2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca^2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca^2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau     

Successful cyclosporine treatment for atopic dermatitis in a rhesus macaque (Macaca mulatta)

A juvenile (1 year old ) female rhesus macaque (Macaca mulatta) developed a chronic active skin condition characterized by pruritus, erythema, alopecia, scaling, exfoliation, and lichenification. Lesions were limited to the ventrum, specifically rostral mandible and neck, axilla and inguinal regions, distal extremities, and interdigital regions. Differential diagnoses included infection, dietary deficiency, metabolic abnormality, endocrinopathy, and immunological injury. Diagnostic tests included complete hemogram, serum chemistry, skin scrapes for ectoparasite detection, hair plucks for dermatophyte culture, and a serum-based hypersensitivity panel. All results were within normal limits. Dermal biopsies revealed lesions consistent with active allergic dermatitis, and a diagnosis of atopic dermatitis was made. Oral cyclosporine (5 mg/kg daily) rapidly eliminated clinical evidence of dermatitis. Histologically, lesions resolved after 12 months of treatment. Atopic dermatitis is an inflammatory skin condition for which there are neither pathognomonic clinical or diagnostic features nor a single successful therapy. Basic criteria such as pruritus, lichenification, a chronic course, and history of allergies strongly support the diagnosis. One successful therapeutic agent is a macrolide calcineurin inhibitor, cyclosporine. It represents a safer class of immunomodulatory drugs than corticosteroids and provides targeted alteration of lymphocyte function. To our knowledge this case represents the first reported successful treatment of atopic dermatitis in a nonhuman primate utilizing cyclosporine.     

Frequency of sister-chromatid exchange among greenhouse farmers exposed to pesticides

Sister-chromatid exchange (SCE) was measured in peripheral lymphocytes of 104 greenhouse farmers exposed to pesticides and 44 unexposed workers. The results of SCEs are expressed in two variables: (a) mean number of SCEs per chromosome and, (b) proportion of high frequency cells (cells with more than eight SCEs). A high correlation was found between these two variables. The adjusted means of both SCEs variables were significantly higher among the farmers compared with the unexposed group (P < 0.01). Adjustment was made for smoking, age, education, and origin.The adjusted means of both SCE variables, were significantly elevated (P < 0.05) among the farmers who prepared and applied more than 70% of the pesticides by themselves compared with those who prepared and applied less than 70% of the pesticides by themselves. Both SCEs variables were also significantly elevated (P < 0.05) among farmers who were involved in more than 7.4 sprays per year compared with those with 7.4 or less sprays per year (P < 0.05). We found a tendency towards elevation of the two variables of SCEs among those who did not use protective measures while preparing the pesticides.Evaluation of the influence of years of exposure on the frequency of SCEs showed that the two variables of SCEs were higher among those farmers who were exposed to pesticides for more than 21 years than among those with less than 21 years of exposure. The variables that had the most influence on the elevation of SCEs were self-preparation of the pesticide mixtures and the number of sprayings per year. Because the farmers used a mixture of almost 24 different chemical classes it was impossible to attribute exposure to a specific pesticide or group of pesticides to single farmers.Our finding of a significant increase of SCEs frequency in peripheral lymphocytes in greenhouse farmers indicates a potential cytogenetic hazard due to pesticides exposure.     

Low Message Sensation Health Promotion Videos Are Better Remembered and Activate Areas of the Brain Associated with Memory Encoding

Greater sensory stimulation in advertising has been postulated to facilitate attention and persuasion. For this reason, video ads promoting health behaviors are often designed to be high in “message sensation value” (MSV), a standardized measure of sensory intensity of the audiovisual and content features of an ad. However, our previous functional Magnetic Resonance Imaging (fMRI) study showed that low MSV ads were better remembered and produced more prefrontal and temporal and less occipital cortex activation, suggesting that high MSV may divert cognitive resources from processing ad content. The present study aimed to determine whether these findings from anti-smoking ads generalize to other public health topics, such as safe sex. Thirty-nine healthy adults viewed high- and low MSV ads promoting safer sex through condom use, during an fMRI session. Recognition memory of the ads was tested immediately and 3 weeks after the session. We found that low MSV condom ads were better remembered than the high MSV ads at both time points and replicated the fMRI patterns previously reported for the anti-smoking ads. Occipital and superior temporal activation was negatively related to the attitudes favoring condom use (see Condom Attitudes Scale, Methods and Materials section). Psychophysiological interaction (PPI) analysis of the relation between occipital and fronto-temporal (middle temporal and inferior frontal gyri) cortices revealed weaker negative interactions between occipital and fronto-temporal cortices during viewing of the low MSV that high MSV ads. These findings confirm that the low MSV video health messages are better remembered than the high MSV messages and that this effect generalizes across public health domains. The greater engagement of the prefrontal and fronto-temporal cortices by low MSV ads and the greater occipital activation by high MSV ads suggest that that the “attention-grabbing” high MSV format could impede the learning and retention of public health messages.     

Design of a basigin-mimicking inhibitor targeting the malaria invasion protein RH5

Many human pathogens use host cell-surface receptors to attach and invade cells. Often, the host-pathogen interaction affinity is low, presenting opportunities to block invasion using a soluble, high-affinity mimic of the host protein. The Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) provides an exciting candidate for mimicry: it is highly conserved and its moderate affinity binding to the human receptor basigin (K_D ≥1 μM) is an essential step in erythrocyte invasion by this malaria parasite. We used deep mutational scanning of a soluble fragment of human basigin to systematically characterize point mutations that enhance basigin affinity for RH5 and then used Rosetta to design a variant within the sequence space of affinity-enhancing mutations. The resulting seven-mutation design exhibited 1900-fold higher affinity (K_D approximately 1 nM) for RH5 with a very slow binding off rate (0.23 h⁻¹) and reduced the effective Plasmodium growth-inhibitory concentration by at least 10-fold compared to human basigin. The design provides a favorable starting point for engineering on-rate improvements that are likely to be essential to reach therapeutically effective growth inhibition.