Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.     

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (T_m) of the mutants over the native BcEST that has a T_m of 50°C was outperformed by TtEST, a naturally occurring homologue with a T_m of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.     

Endocardial Tip Cells in the Human Embryo – Facts and Hypotheses

Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43–56 days) were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin), CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.     

Interspecies Gene Name Extrapolation—A New Approach

The use of animal models has facilitated numerous scientific developments, especially when employing “omics” technologies to study the effects of various environmental factors on humans. Our study presents a new bioinformatics pipeline suitable when the generated microarray data from animal models does not contain the necessary human gene name annotation. We conducted single color gene expression microarray on duodenum and spleen tissue obtained from pigs which have been exposed to zearalenone and Escherichia coli contamination, either alone or combined. By performing a combination of file format modifications and data alignments using various online tools as well as a command line environment, we performed the pig to human gene name extrapolation with an average yield of 58.34%, compared to 3.64% when applying more simple methods. In conclusion, while online data analysis portals on their own are of great importance in data management and assessment, our new pipeline provided a more effective approach for a situation which can be frequently encountered by researchers in the “omics” era.     

High incidence of ubiquitin-like domains in human ubiquitin-specific proteases

Ubiquitin-specific proteases (USPs) emerge as key regulators of numerous cellular processes and account for the bulk of human deubiquitinating enzymes (DUBs). Their modular structure, mostly annotated by sequence homology, is believed to determine substrate recognition and subcellular localization. Currently, a large proportion of known human USP sequences are not annotated either structurally or functionally, including regions both within and flanking their catalytic cores. To extend the current understanding of human USPs, we applied consensus fold recognition to the unannotated content of the human USP family. The most interesting discovery was the marked presence of reliably predicted ubiquitin-like (UBL) domains in this family of enzymes. The UBL domain thus appears to be the most frequently occurring domain in the human USP family, after the characteristic catalytic domain. The presence of multiple UBL domains per USP protein, as well as of UBL domains embedded in the USP catalytic core, add to the structural complexity currently recognized for many DUBs. Possible functional roles of the newly uncovered UBL domains of human USPs, including proteasome binding, and substrate and protein target specificities, are discussed.     

Structural aspects of recently discovered viral deubiquitinating activities

Protein ubiquitination has been identified as a regulatory mechanism in key cellular activities, and deubiquitination is recognized as an important step in processes governed by ubiquitin and ubiquitin-like modifiers. Viruses are known to target ubiquitin and ubiquitin-like modifier pathways using various strategies, including the recruitment of host deubiquitinating enzymes. Deubiquitinating activities have recently been described for proteins from three different virus families (adenovirus, coronavirus and herpesvirus), and predicted for others. This review centers on structural-functional aspects that characterize the confirmed viral deubiquitinating enzymes, and their relationships to established families of cellular deubiquitinating enzymes.     

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Lateral organization of biological membranes is frequently studied using fluorescence microscopy. One of the most widely used probes for these studies is 2-dimethylamino-6-lauroylnaphthalene (laurdan). The fluorescence of this probe is sensitive to the environment polarity, and thus laurdan reports the local penetration of water when inserted in membranes. Unfortunately, this probe can only be used under two-photon excitation due to its low photostability. This is a very important limitation, because there are not too many laboratories with capability for two-photon microscopy. In this work, we explored the performance of 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), a carboxyl-modified version of laurdan, for imaging biological membranes using a conventional confocal microscopy setup. We acquired generalized polarization (GP) images of C-laurdan inserted in giant unillamelar vesicles composed of binary mixtures of lipids and verified that the probe allows observing the coexistence of different phases. We also tested the performance of the probe for measurement with living cells and registered GP images of melanophore cells labeled with C-laurdan in which we could observe highly ordered regions such as filopodia. These findings show that C-laurdan can be successfully employed for studies of membrane lateral organization using a conventional confocal microscope and can open the possibility of studying a wide variety of membrane-related processes.     

Structure and active site residues of PglD, an N-acetyltransferase from the bacillosamine synthetic pathway required for N-glycan synthesis in Campylobacter jejuni

Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2,4-diacetamido-2,4,6-trideoxy-d-Glc (QuiNAc4NAc or N,N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2,4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 A resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal alpha/beta-domain and a C-terminal left-handed beta-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the beta-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the beta-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an "L-shape", compared with the "in-line" orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.     

Caveolin-1 overexpression correlates with tumour progression markers in pancreatic ductal adenocarcinoma

The assessment of caveolin-1 (Cav-1) as a marker of tumor aggressiveness in pancreatic ductal adenocarcinoma (PDAC). In this study, we examined the expression of Cav-1 in 34 human PDAC tissue samples and the associated peritumoral tissues by immunohistochemistry and western blot. Additionally, we correlated Cav-1 expression with other tissue (Ki-67, p53) and serum (CA 19-9) tumor markers. In the tumor-derived tissue, both tumor cells and blood vessels expressed Cav-1. In contrast, in peritumoral tissue, Cav-1 expression was confined mainly to blood vessels and was only occasionally expressed in ductal or parenchymal cells. Western blot analysis confirmed the overexpression of Cav-1 in pancreatic tumors compared with peritumoral tissue. Cav-1 expression in tumor tissues was correlated with both the Ki-67 LI (r = 0.95, P < 0.0001) and p53 expression (χ2 = 9.91, P < 0.005). Overexpression of Cav-1 was associated with tumor size, grade and stage and Cav-1 expression in tumors was correlated with an increased serum level of CA 19-9 (r = 0.795, P < 0.001). Based on the results of this study, the inclusion of Cav-1 in a putative panel of biomarkers predicting pancreatic cancer aggressiveness is warranted.     

Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence -->3)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-d-GlcNAc-(1-->. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstrom resolution and bound to acetyl-CoA at a 1.66-Angstrom resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.