Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17

Metalloproteases play a complex role in tumor progression. While the activity of some ADAM, ADAMTS and matrix metalloproteases (MMPs) seems to be protumorigenic, the activity of others seems to prevent tumor progression. The identification of the array of substrates of a given metalloprotease (degradome) seems an adequate approach to predict the effect of the inhibition of a metalloprotease in tumors. Here, we present the proteomic identification of a novel substrate for ADAM10 and -17. We used SILAC (Stable Isotope Labeling by Amino acids in Cell culture), a proteomic technique based on the differential metabolic labeling of cells in different conditions. This was applied to MCF7 cells derived from an invasive mammary tumor, and the same cells expressing shRNAs that knock down ADAM10 or -17. Following this approach, we have identified C4.4A as a substrate to both metalloproteases. Since C4.4A is likely involved in tumor invasion, these results indicate that the cleavage of C4.4A by ADAM10 and ADAM17 contributes to tumor progression.     

Prestin's role in cochlear frequency tuning and transmission of mechanical responses to neural excitation

The remarkable power amplifier [1] of the cochlea boosts low-level and compresses high-level vibrations of the basilar membrane (BM) [2]. By contributing maximally at the characteristic frequency (CF) of each point along its length, the amplifier ensures the exquisite sensitivity, narrow frequency tuning, and enormous dynamic range of the mammalian cochlea. The motor protein prestin in the outer hair cell (OHC) lateral membrane is a prime candidate for the cochlear power amplifier [3]. The other contender for this role is the ubiquitous calcium-mediated motility of the hair cell stereocilia, which has been demonstrated in vitro and is based on fast adaptation of the mechanoelectrical transduction channels [4, 5]. Absence of prestin [6] from OHCs results in a 40-60 dB reduction in cochlear neural sensitivity [7]. Here we show that sound-evoked BM vibrations in the high-frequency region of prestin(-/-) mice cochleae are, surprisingly, as sensitive as those of their prestin(+/+) siblings. The BM vibrations of prestin(-/-) mice are, however, broadly tuned to a frequency approximately a half octave below the CF of prestin(+/+) mice at similar BM locations. The peak sensitivity of prestin(+/+) BM tuning curves matches the neural thresholds. In contrast, prestin(-/-) BM tuning curves at their best frequency are >50 dB more sensitive than the neural responses. We propose that the absence of prestin from OHCs, and consequent reduction in stiffness of the cochlea partition, changes the passive impedance of the BM at high frequencies, including the CF. We conclude that prestin influences the cochlear partition's dynamic properties that permit transmission of its vibrations into neural excitation. Prestin is crucial for defining sharp and sensitive cochlear frequency tuning by reducing the sensitivity of the low-frequency tail of the tuning curve, although this necessitates a cochlear amplifier to determine the narrowly tuned tip.     

Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated with contrasting levels of nitrogen fertilizer.

The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres, and the PCR products cloned and sequenced. Four clone libraries were generated from the nifH fragments and 245 sequences were obtained. Most of the clones (57%) were closely related to nifH genes of uncultured bacteria. NifH clones affiliated with Azohydromonas spp., Ideonella sp., Rhizobium etli and Bradyrhizobium sp. were found in all libraries. Sequences affiliated with Delftia tsuruhatensis were found in the rhizosphere of both cultivars sown with high levels of nitrogen, while clones affiliated with Methylocystis sp. were detected only in plants sown under low levels of nitrogen. Moreover, clones affiliated with Paenibacillus durus could be found in libraries from the cultivar IS 5322-C sown either in high or low amounts of fertilizer. This study showed that the amount of nitrogen used for fertilization is the overriding determinative factor that influenced the nitrogen-fixing community structures in sorghum rhizospheres cultivated in Cerrado soil.     

Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the β1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that αVβ3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.     

Contribution of the Periplasmic Chaperone Skp to Efficient Presentation of the Autotransporter IcsA on the Surface of Shigella flexneri

IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane β-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the β-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the β-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.Type V secretion apparatuses (also called autotransporters) consist of an extensive class of large, outer membrane proteins of gram-negative bacteria, typically virulence factors, found in all subdivisions of proteobacteria (28). Although originally designated as “autotransporters” because they were thought to mediate their own insertion into and translocation across the outer membrane, more recent evidence suggests that autotransporter secretion and insertion requires the aid of accessory factors (21, 29). Secretion involves the insertion of the carboxy-terminal β-barrel domain into the outer membrane and translocation of the mature passenger (alpha) domain across the outer membrane (Fig. ​(Fig.1).1). Whether these two events occur sequentially or simultaneously is unclear. Analysis of crystal structures indicates that the carboxy-terminal end of the passenger domain is present within the central pore of the β-barrel (4, 27). Several studies provide evidence that at least some autotransporters are partially folded in the periplasm (7, 20), and one of these studies provides strong evidence that the passenger domain may be partially or fully incorporated into the β-barrel prior to incorporation of the mature protein into the outer membrane (20).Open in a separate windowFIG. 1.Schematic of the autotransporter IcsA. (A) Linear diagram showing the signal peptide (SP), alpha-domain (IcsA_53-757), and carboxy-terminal β-barrel domain. (B) IcsA in the outer membrane. The carboxy-terminal β-barrel is inserted into the outer membrane, and the mature amino-terminal alpha-domain is exposed on the bacterial surface. N′, mature amino terminus; C, carboxyl terminus; OM, outer membrane; arrow, proposed site of cleavage between residues 757 and 758 by IcsP.Shigella flexneri is a gram-negative human pathogen which, upon passage through the lower digestive tract, gains entry into colonic epithelial cells. Once S. flexneri is intracellular, it spreads to adjacent cells by secreting IcsA, a surface-associated autotransporter that is required for the polymerization of host cell actin on the bacterial surface. Actin polymerization occurs at a single pole of the bacterium and is required for infection of adjacent cells and disease pathogenesis (5, 24, 33).IcsA is encoded on a large virulence plasmid. The full-length protein is approximately 120 kDa and has three assigned functional and structural domains (25): an atypical Sec secretion signal (IcsA_1-52), the alpha-domain (IcsA_53-757), which is exposed on the bacterial surface and contains sequences that are required for actin polymerization, and the beta-domain (IcsA_758-1102), which forms a β-barrel structure in the outer membrane (Fig. ​(Fig.1A)1A) (21, 25). In vivo, a fraction of IcsA molecules are proteolytically processed at the junction between the alpha- and beta-domains by the protease IcsP (SopA), a protein which is also encoded on the virulence plasmid (14, 34). IcsA_53-757 is found in the supernatant of liquid cultures, while mature full-length IcsA (IcsA_53-1102), IcsA_758-1102 (14, 34), and some IcsA_53-757 (this work) remain cell associated. IcsA, like other autotransporters, is secreted at the bacterial pole (22), the site at which actin tail assembly occurs. As it is for other β-barrel-containing outer membrane proteins, insertion of IcsA and other autotransporters into the outer membrane requires the outer membrane insertase YaeT (BamA, Omp85) (21).Skp, DegP, and SurA are periplasmic chaperones that, like YaeT, appear to function in the targeting and/or insertion of outer membrane proteins (35). Evidence based on synthetic phenotypes suggests that during outer membrane protein insertion Skp and DegP act in one pathway and that SurA acts in a distinct but parallel pathway (35).We investigated the role of the periplasmic chaperone Skp in the folding and secretion of IcsA in S. flexneri. We found that in the absence of skp, IcsA is inefficiently presented on the surface of S. flexneri, leading to a cellular spread defect. Surprisingly, the protein was still efficiently cleaved by the outer membrane protease IcsP, as wild-type levels of IcsA_53-757 were detected in the culture supernatants. We found that introduction of the icsP mutation into the skp strain background led to an increase in the levels of full-length IcsA presented on the bacterial cell surface of the skp mutant, and we present models that could explain our results.     

Mutations in Smooth Muscle Alpha-Actin (ACTA2) Cause Coronary Artery Disease, Stroke, and Moyamoya Disease, Along with Thoracic Aortic Disease

The vascular smooth muscle cell (SMC)-specific isoform of α-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.     

Trace Element Composition of Selected Fertilizers Used in Chile: Phosphorus Fertilizers as a Source of Long-Term Soil Contamination

Anthropogenic activities like agriculture have resulted in increased concentrations of some trace elements of toxicological and environmental concern in soils. Application of fertilizers has been one of the major inputs of these contaminants to agricultural soils in developing countries. Twenty-two fertilizers, including straight nitrogen (N), phosphorus (P), potassium (K), and NK fertilizers and micronutrient sources, were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) for arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn). As expected, the trace element content of fertilizers was highly variable and related to the origin of the material. Phosphorus fertilizers, especially triple superphosphate, presented the highest As, Cd, Cu, Cr, Ni, V, and Zn concentrations. In some of these fertilizers, the Cr, V, and Zn contents reached values greater than 3475 mg kg^?1 of P, and the Cd content (up to 288 mg kg^?1 of P) was several times higher than the regulatory limits of different countries. Some micronutrient sources presented the highest concentrations of Mn and Pb. In the cases of N, K, and NK fertilizers, the trace element concentration was very low, sometimes below the detection limits. In some agricultural systems the input of trace elements such as As and Cd to the soil through P fertilizers application may be higher than the outputs through plant uptake and leaching; therefore the long-term use of these fertilizers may cause the trace element concentration to increase in the plow layer of agricultural soils.