pH-dependent binding of the Epsin ENTH domain and the AP180 ANTH domain to PI(4,5)P2-containing bilayers

Epsin and AP180 are essential components of the endocytotic machinery, which controls internalization of protein receptors and other macromolecules at the cell surface. Epsin and AP180 are recruited to the plasma membrane by their structurally and functionally related N-terminal ENTH and ANTH domains that specifically recognize PtdIns(4,5)P₂. Here, we show that membrane anchoring of the ENTH and ANTH domains is regulated by the acidic environment. Lowering the pH enhances PtdIns(4,5)P₂ affinity of the ENTH and ANTH domains reinforcing their association with lipid vesicles and monolayers. The pH dependency is due to the conserved histidine residues of the ENTH and ANTH domains, protonation of which is necessary for the strong PtdIns(4,5)P₂ recognition, as revealed by liposome binding, surface plasmon resonance, NMR, monolayer surface tension and mutagenesis experiments. The pH sensitivity of the ENTH and ANTH domains is reminiscent to the pH dependency of the FYVE domain suggesting a common regulatory mechanism of membrane anchoring by a subset of the PI-binding domains.     

Proteolysis of the membrane type-1 matrix metalloproteinase prodomain: implications for a two-step proteolytic processing and activation

Membrane type-1 matrix metalloproteinase (MT1-MMP) exerts its enhanced activity in multiple cancer types. Understanding the activation process of MT1-MMP is essential for designing novel and effective cancer therapies. Like all of the other MMPs, MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its inhibitory prodomain. Proteolytic processing of the prodomain transforms the zymogen into a catalytically active enzyme. A sequential, two-step activation process is normally required for MMPs. Our in silico modeling suggests that the prodomain of MT1-MMP exhibits a conserved three helix-bundled structure and a "bait" loop region linking helixes 1 and 2. We hypothesized and then confirmed that in addition to furin cleavage there is also a cleavage at the bait region in the activation process of MT1-MMP. A two-step sequential activation of MT1-MMP is likely to include the MMP-dependent cleavage at either P47GD downward arrowL50 or P58QS downward arrowL61 or at both sites of the bait region. This event results in the activation intermediate. The activation process is then completed by a proprotein convertase cleaving the inhibitory prodomain at the R108RKR111 downward arrowY112 site, where Tyr112 is the N-terminal residue of the mature MT1-MMP enzyme. Our findings suggest that the most efficient activation results from a two-step mechanism that eventually is required for the degradation of the inhibitory prodomain and the release of the activated, mature MT1-MMP enzyme. These findings shed more light on the functional role of the inhibitory prodomain and on the proteolytic control of MT1-MMP activation, a crucial process that may be differentially regulated in normal and cancer cells.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.     

Domain I of ribosomal protein L1 is sufficient for specific RNA binding

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.     

Genetic diversity of 15 STR loci in a population of Montenegro

Genetic diversity and forensic parameters based on 15AmpFlSTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) were evaluated in a sample of 101 unrelated, autochthonous adults from Montenegro. After applying Bonferroni correction, the agreement with Hardy-Weinberg equilibrium (HWE) was confirmed for all loci with the exception of D5S818 (chi2 test) and D21S11 (exact test). The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 15 studied loci were 0.9999999999999999844 and 0.99999382, respectively. According to measures of within-population genetic diversity, D2S1338, D18S51 and FGA may be considered as the most variable and most informative markers for forensic testing and population genetic analyses out of the 15 analysed loci in a population of Montenegro. D5S818 showed to be the least variable and together with TPOX, the least informative. Interpopulation comparisons were carried out and levels of genetic differentiation between population of Montenegro and five South-eastern European populations (Kosovo Albanians, Serbians from Vojvodina province, Macedonians, Bosnians and Croatians) were evaluated. The most differentiated population in relation to Montenegro is a population of Kosovo Albanians as suggested by both AMOVA and coefficients of genetic differentiation (F(ST) and R(ST)).     

Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship

Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI's of 5.4, of 5.2, of 4.9 and of 4.7. Surprisingly silicatein not only mediates polymerization of silicate, but also displays proteolytic activity which is specific for cathepsin L enzymes, thus underscoring the high relationship of the silicateins to cathepsin L. The cDNAs from L. baicalensis for silicatein and cathepsin L, as well as the respective genes, were cloned. It was found that the five introns present in the sponge genes are highly conserved up to human cathepsin L. This analysis has been completed by sequencing of two silicatein genes (both for silicatein-alpha and -beta) and of cathepsin L from another demosponge, Suberites domuncula. A comprehensive phylogenetic analysis with these new sequences shed new light upon the evolution of cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsin L.     

Seasonal habitat use of three predatory fishes in a freshwater ecosystem

Hydrobiologia - To understand the spatiotemporal overlap in the habitat use of sympatric predators, we studied longitudinal activity and reservoir section and depth use of pike (Esox lucius),...     

Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.     

Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging

Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via 'over-clocking' of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in ～24 h.