Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.

Dynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic schemewhere k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.Keywords: horeseradish peroxidase; differential scanning calorimetry; intrinsic fluorescence; circular dichroism; irreversible denaturation.Horseradish peroxidase (HRP) belongs to the superfamily of the heme-containing plant peroxidases (EC 1.11.1.7), which has been divided into three classes [1], supported in the first instance by comparison of amino-acid sequence data and confirmed by more recent data on crystal structures [2]. Plant peroxidases, including HRP, comprise class III of the superfamily. Although the function of peroxidases is often seen primarily in terms of the conversion of H 2 O 2 to H 2 O, this should not be allowed to mask their wider participation in other reactions, many of which are biologically significant. Despite the enormous interest in peroxidases owing to their broad practical applications in biotechnology, the data concerning their structural stability are sparse. Although several publications have addressed the thermal stability of peroxidases [3±8], to date the mechanism of the process of thermal denaturation remains unclear. It is known that the biological functions of proteins depend on the correct folding of their native structure and that loss of this folded structure leads to an unfolded, inactive state. Consequently, the study of protein stability is important both from the academic and applied points of view.Factors affecting conformational stability have been studied most intensively in proteins under reversible conditions [9±15]. Nevertheless, it is well known that for different reasons many proteins cannot refold in vitro after denaturation such as proteolytic digestion [16], aggregation, loss of prosthetic group, the cis/trans izomerization of certain proline residues [17,18] or chemical modifications [19]. Generally, the thermal denaturat...

The HIV-1 gp41 envelope protein mediates entry of the virus into the target cell by promoting membrane fusion. With a view toward possible new insights into viral fusion mechanisms, we have investigated by infrared, fluorescence, and nuclear magnetic resonance spectroscopies and calorimetry a fragment of 19 amino acids corresponding to the immunodominant region of the gp41 ectodomain, a highly conserved sequence and major epitope. Information on the structure of the peptide both in solution and in the presence of model membranes, its incorporation and location in the phospholipid bilayer, and the modulation of the phase behavior of the membrane has been gathered. Here we demonstrate that the peptide binds and interacts with negatively charged phospholipids, changes its conformation in the presence of a membraneous medium, and induces leakage of vesicle contents as well as a new phospholipid phase. These characteristics might be important for the formation of the fusion-active gp41 core structure, promoting the close apposition of the two viral and target-cell membranes and therefore provoking fusion.

Recent data from multiple laboratories indicate that upon infection, many di¡erent families of viruses hijack the dynein motor machinery and become transported in a retrograde manner towards the cell nucleus. In certain cases, one of the dynein light chains, LC8, is involved in this interaction. Using a library of overlapping dodecapeptides synthesized on a cellulose membrane (pepscan technique) we have analyzed the interaction of the dynein light chain LC8 with 17 polypeptides of viral origin. We demonstrate the strong binding of two herpesvirus polypeptides, the human adenovirus protease, vaccinia virus polymerase, human papillomavirus E4 protein, yam mosaic virus polyprotein, human respiratory syncytial virus attachment glycoprotein, human coxsackievirus capsid protein and the product of the AMV179 gene of an insect poxvirus to LC8. Our data corroborate the manipulation of the dynein macromolecular complex of the cell during viral infection and point towards the light chain LC8 as one of the most frequently used targets of virus manipulation. ß

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Sitedirected mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and ⅐NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker ␤-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the ⅐NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization. The gaseous radical nitric oxide (⅐NO)1 modulates biological function in a wide range of tissue types, acting either as a signaling molecule or as a toxin. Three human NOS isoforms have been cloned and characterized. Among them, NOS2(sometimes referred to as inducible NOS or iNOS) is mostly involved in the synthesis of the large amounts of ⅐NO that appear in inflammatory and immunologic processes (1, 2).Both crystallographic and enzymatic studies performed with recombinant proteins expressed in Escherichia coli have shown that the N terminus end of the three mammalian NOSs is not involved in ⅐NO synthesis but rather in subcellular targeting of the mature polypeptide chain (2, 3). For instance, the PDZ domain of NOS1 (residues 1-90) interacts with dystrophin and becomes localized to the sarcolemma of fast twitch fibers (4). In fact, deletion of the first 226 amino acids of NOS1 results in a catalytic protein that synthesizes ⅐NO at a similar rate than the full-length protein (5). Likewise, the N-terminal end of NOS3 is covalently and irreversibly myristoylated at Gly-2 and reversibly palmitoylated at Cys-15 and Cys-26 in a well described process responsible for its targeting to caveolae (6, 7). In addition, deletion of the first 52 amino acids of NOS3 does not affect catalytic activity, reflecting that this sequence stretch is not part of the enzymatic machinery but is involved in intrac...