Volume 385, Issue 2

The breakdown of the barriers formed by extracellular matrix proteins is a pre-requisite for all processes of tissue remodeling. Matrix degradation reactions take part in specific physiological events in the healthy organism but also represent a crucial step in cancer invasion. These degradation processes involve a highly organized interplay between proteases and their cellular binding sites as well as specific substrates and internalization receptors. This review article is focused on two components, the urokinase plasminogen activator receptor (uPAR) and the uPAR-associated protein (uPARAP, also designated Endo180), that are considered crucially engaged in matrix degradation. uPAR and uPARAP have highly diverse functions, but on certain cell types they interact with each other in a process that is still incompletely understood. uPAR is a glycosyl-phosphatidylinositol-anchored glycoprotein on the surface of various cell types that serves to bind the urokinase plasminogen activator and localize the activation reactions in the proteolytic cascade system of plasminogen activation. uPARAP is an integral membrane protein with a pronounced role in the internalization of collagen for intracellular degradation. Both receptors have additional functions that are currently being unraveled. The present discussion of uPAR and uPARAP is centered on their protein structure and molecular and cellular function.

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep -tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 å resolution.

The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate 7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotidesugar level [Shen et al., Biol. Chem. 383 (2002), 307317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMPNeu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMPNeu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-Nacetyl-9-O-acetylneuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-Nacetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMPNeu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galβ1,3(4)R- residues.

The intrinsic apoptosis apparatus plays a significant role in generating and amplifying cell death signals. In this study we examined whether there are differences in the expression of its components and in its functioning in nonsmall cell lung carcinoma (NSCLC) and the lung. We show that NSCLC cell lines express Apaf-1 and procaspase-9 and -3 proteins and that the expression of Apaf-1 and procaspase-3, but not of procaspase-9 and -7, is frequently upregulated in NSCLC tissues as compared to the lung. NSCLC tissues and lungs and some NSCLC cell lines expressed also caspase-9S(b) and displayed a high caspase-9S(b)/procaspase-9 expression ratio. Procaspase-3 from NSCLCs and lungs was readily processed to caspase-3 by granzyme B or caspase-8, and the granzyme Bgenerated caspase-3-like activity was significantly higher in tumor tissues and cells than in lungs. By contrast, cytochrome c plus dATP could induce a significant increase of caspase-3-like activity in cytosol only in some NSCLC cell lines and in subsets of studied NSCLC tissues and lungs, while procaspase-3 and -7 were detectably processed only in NSCLC tissues which showed a high (cytochrome c+dATP)-induced caspase-3-like activity. Taken together, the present study provides evidence that the expression of Apaf-1 and procaspase 3 is up-regulated in NSCLCs and indicates that the tumors have a capability to suppress the apoptosome-driven caspase activation in their cytosol.

The hormone glucagon-like peptide-1(736)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucose-dependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the ?amino group in the side chain of the Lys26 residue and to combine this modification with substitutions of the Ala8 residue, namely Val or aminobutyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys(pal)26]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal ?cell line BRINBD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRINBD11 cells was similar, or in the case of [Val8,Lys(pal)26]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)26]GLP-1, [Abu8,Lys(pal)26]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucoselowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.

Recent reports presented contradictory results regarding the catabolism of mature atrial (ANP) and brain (BNP) natriuretic peptides in circulation. Especially the role of neutral endopeptidase (NEP) in BNP degradation was conversely discussed. Our present in vitro -studies characterize the NEP-dependent metabolism of ANP and BNP in different tissues via HPLC-analysis using NEP-deficient mice and specific NEP inhibitors. Our results show a strong tissue-dependent degradation pattern of both peptides, which are not only due to the different NEP activities in these tissues. Whereas NEP rapidly degraded ANP, it had no influence in BNP-metabolism. Additional experiments with purified NEP confirmed this result. Moreover, we describe a degradation of ANP and BNP in NEP-deficient and NEP-inhibited membranes. Consequently, we postulate the existence of at least one further natriuretic peptide (NP) degrading enzyme, which has not been characterized yet. Thus, the commonly accepted model of the natriuretic peptide system with NEP as the central degrading peptidase has to be partly revised. Moreover, the NEP-independent BNP degradation provides an effective means for achieving a beneficial BNP increase in cardiovascular pathology by inhibiting the assumed novel NP-degrading peptidase(s).

A 7514-Da chymotrypsin inhibitor was isolated from the seed extract of Momordica cochinchinensis (Family Cucurbitaceae) by chromatography on chymotrypsin Sepharose 4B and subsequently by C18 reversed-phase HPLC. This inhibitor, named MCoCI, possessed remarkable thermostability and was stable from pH 2 to 12. MCoCI also inhibited subtilisin, but had at least 50-fold lower inhibitory activity towards trypsin and elastase. Amino acid sequencing of a peptide fragment of MCoCI revealed a sequence of 23 amino acids. Comparison of this sequence and the molecular mass with those of other protease inhibitors suggests that MCoCI belongs to the potato I inhibitor family.

The expanded genetic code in combination with sitedirected mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange (atomic mutation) and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphuraromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.