CATH--a hierarchic classification of protein domain structures

BACKGROUND: Protein evolution gives rise to families of structurally related proteins, within which sequence identities can be extremely low. As a result, structure-based classifications can be effective at identifying unanticipated relationships in known structures and in optimal cases function can also be assigned. The ever increasing number of known protein structures is too large to classify all proteins manually, therefore, automatic methods are needed for fast evaluation of protein structures. RESULTS: We present a semi-automatic procedure for deriving a novel hierarchical classification of protein domain structures (CATH). The four main levels of our classification are protein class (C), architecture (A), topology (T) and homologous superfamily (H). Class is the simplest level, and it essentially describes the secondary structure composition of each domain. In contrast, architecture summarises the shape revealed by the orientations of the secondary structure units, such as barrels and sandwiches. At the topology level, sequential connectivity is considered, such that members of the same architecture might have quite different topologies. When structures belonging to the same T-level have suitably high similarities combined with similar functions, the proteins are assumed to be evolutionarily related and put into the same homologous superfamily. CONCLUSIONS: Analysis of the structural families generated by CATH reveals the prominent features of protein structure space. We find that nearly a third of the homologous superfamilies (H-levels) belong to ten major T-levels, which we call superfolds, and furthermore that nearly two-thirds of these H-levels cluster into nine simple architectures. A database of well-characterised protein structure families, such as CATH, will facilitate the assignment of structure-function/evolution relationships to both known and newly determined protein structures.     

Detection of blotted antigen using a fusion protein between protein A and pepsinogen C

Human pepsinogen (PG) A and C were fused with protein A and expressed in Escherichia coli. Although the fusion proteins (PA-PGA and PA-PGC) were not expressed at high levels and were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. PA-PGA and PA-PGC possessed proteolytic activity equivalent to the gastric mucosal PGA and PGC, respectively. However, the activity of PA-PGC was about 3-fold higher than that of PA-PGA. Therefore, PA-PGC was applied to the subsequent immunoblotting studies. The proteolytic activity of PA-PGC was used for digesting the blocking reagent around the target antigen (in situ digestion method) or casein-clotting in the agarose plate containing skimmed milk (caseogram print method). Although the sensitivity of these methods was lower than that of the conventional color detection, the caseogram print method was superior in that the reaction was linear over a wide range. On the other hand, the in situ digestion method possessed a unique property on Western blotting, and it was very easy to identify the relative position of the target, which could be recognized as a clear band. For PA-PGC detection, no special chemicals are required, and so the procedure is simple, rapid, and inexpensive.     

Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases

The suppression and eradication of primary tumors and distant metastases is a major goal of alternative treatment strategies for cancer, such as inhibition of angiogenesis and targeted immunotherapy. We report here a synergy between two novel monotherapies directed against vascular and tumor compartments, respectively, a tumor vasculature-specific antiangiogenic integrin alphav antagonist and tumor-specific antibody-interleukin 2 (IL-2) fusion proteins. Simultaneous and sequential combination of these monotherapies effectively eradicated spontaneous liver metastases in a poorly immunogenic syngeneic model of neuroblastoma. This was in contrast to controls subjected to monotherapies with either an antiangiogenic integrin alphav antagonist or antibody-IL-2 fusion proteins, which were only partially effective at the dose levels applied. Furthermore, simultaneous treatments with the integrin alphav antagonist and tumor-specific antibody-IL-2 fusion proteins induced dramatic primary tumor regressions in three syngeneic murine tumor models, i.e., melanoma, colon carcinoma, and neuroblastoma. However, each agent used as monotherapy induced only a delay in tumor growth. A mechanism for this synergism was suggested because the antitumor response was accompanied by a simultaneous 50% reduction in tumor vessel density and a 5-fold increase in inflammatory cells in the tumor microenvironment. Subsequently, tumor necrosis was demonstrated only in animals receiving the combination therapy, but not when each agent was applied as monotherapy. The results suggest that these synergistic treatment modalities may provide a novel and effective tool for future therapies of metastatic cancer.     

A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein

Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity. The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography. This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338). The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding. The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8. These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8. Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion. Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC.     

Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells

Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.     

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Bacterial biofilm formation on contact lenses (CLs), and CL storage cases may be a risk factor for CL-associated corneal infection and may explain the persistence of organisms in CL storage cases. This study evaluated biofilm formation on, and microbial contamination of, CLs and CL storage cases from patients with microbial keratitis. Contact lenses and CL storage cases from 20 wearers with microbial keratitis were sampled microbiologically and visualized using scanning electron microscopy (SEM). Culture results from the cornea were also noted. Bacterial biofilm was present more frequently (P < 0.05) on CL storage case surfaces (17/20) compared with CL surfaces (11/20) and biofilm density was significantly greater on case surfaces (P < 0.05). There was no association between poor compliance and microbial contamination of the CL storage case, nor between poor compliance and biofilm formation or density on the CL or CL storage case. Biofilm formation occurred equally frequently with hydrogen peroxide and chlorine release care systems. Microbial keratitis in CL wearers is frequently associated with bacterial biofilm in the CL storage case. Despite the use of current CL disinfection systems, the CL storage case is a favourable environment for proliferation of certain organisms. Biofilm on CLs may prolong the retention time of organisms at the ocular surface and increase their potential pathogenicity.     

Self-association of LIM-kinase 1 mediated by the interaction between an N-terminal LIM domain and a C-terminal kinase domain

LIM-kinase 1 (LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMKI revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.     

Induction of autoantibody responses to GM-CSF by hyperimmunization with an Id-GM-CSF fusion protein

Fusion proteins consisting of an Ig containing xenogenic constant regions and granulocyte-macrophage colony stimulating factor (Id-GM-CSF) are potent immunogens capable of inducing anti-idiotypic Abs after two immunizations, without the usual need for adjuvants or carrier proteins. In this study, we investigated the effects of hyperimmunization with Id-GM-CSF and found that it induces anti-GM-CSF Abs that could bind to GM-CSF and neutralize its bioactivity in vitro. However, no detrimental effects of the anti-GM-CSF activity were apparent on the general health of the animals or on their base line white blood cell counts. Mice with the anti-GM-CSF activity reconstituted their peripheral white blood cells with identical kinetics as control mice after high dose cyclophosphamide treatment, sublethal irradiation, or lethal irradiation followed by syngeneic bone marrow transplantation. Primary and secondary Ab responses to a variety of protein Ags, including an unrelated Ig Id, were not affected. However, the anti-Id response induced by an unrelated GM-CSF fusion protein that is dependent upon the GM-CSF bioactivity was impaired. To avoid any potential problems associated with inducing anti-GM-CSF Abs, we show that priming with the Id-GM-CSF protein and boosting with the Id protein alone were sufficient to induce comparable anti-Id titers without inducing anti-GM-CSF Abs. We conclude that although hyperimmunization of mice with the GM-CSF fusion protein induced neutralizing anti-GM-CSF Abs, this was of little consequence to the animals. Nevertheless, we have devised a strategy to overcome this potential limitation on the use of GM-CSF fusion proteins for immunization.     

Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions

The Lasp-1 gene, which has been localized to the q12-q21 region of human chromosome 17, is amplified and overexpressed in human breast cancers. In addition to the previously reported LIM and SH3 domains of Lasp-1, we report here the identification of an actin-binding domain in the core of the protein. This domain is functional as we demonstrate that Lasp-1 binds actin in vivo and in vitro. In addition, confocal analysis of the Lasp-1 subcellular distribution shows that the protein is colocalized with actin at peripheral cell extensions in individual epithelial cancer cells and in transformed fibroblastic cells. Moreover, Lasp-1 is tyrosine phosphorylated in fibroblast cell lines transformed by a constitutively active form of c-Src (c-SrcY527F). Altogether, our results show that Lasp-1 defines a new type of actin-binding protein and suggest that the protein may play a role in a signaling pathway involved in the organization of the cytoskeleton.     

Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.     

The MBP fusion protein restores the activity of the first phosphatase domain of CD45

Thirty samples of paddy rice and twenty-five of milled rice were obtained from processing centers located in two northern Provinces of Argentina and one southern Province of Paraguay. Contaminating fungi were enumerated by direct plating on dichloran rose bengal chloramphenicol agar and oxytetracycline glucose yeast extract agar before and after surface disinfection. All fungi were isolated and identified to the genus level and percentage infection of samples calculated. Those belonging to the genera Penicillium, Aspergillus and Fusarium were identified to species level. The surface mycoflora was dominated by storage fungi, notably Penicillium citrinum (73% of samples), P. islandicum (60% of samples), Aspergillus niger, A. flavus and Fusarium semitectum. The major fungi found as internal contaminants of paddy rice were, again, Penicillium citrinum (66% of samples) and P. islandicum (50% of samples). Milled rice showed a lower level of contamination, but with a similar species distribution, Penicillium citrinum and P. islandicum again being the main contaminants. The presence of these species suggests a potential for mycotoxin production. Further studies are needed to establish the mycotoxin quality of rice from this region.     

A fluorescent p53GFP fusion protein facilitates its detection in mammalian cells while retaining the properties of wild-type p53

Tumor progression is often characterized by the cumulative loss of crucial cell cycle control genes and the concomitant loss of genome stability. Progressed tumors are often resistant to conventional therapies. Gene-transfer of key growth-regulatory genes, such as the p53 gene, is one potential approach to treating advanced tumors. To this end, we have produced high-titer retroviruses, based on the pCL vector system, which encode a chimeric protein consisting of human wild-type p53 and the green fluorescent protein (wtp53GFP). The fluorescent wtp53GFP protein and the wild-type p53 protein are recognized equally by several monoclonal p53-specific antibodies, have similar half-lives and function comparably in transactivating a p53-responsive element as well as in suppressing the growth of tumor cells. Additionally, due to its fluorescent nature, wtp53GFP facilitates the direct identification of cells expressing the p53 fusion protein. Combining the features of the pCL retroviral production system with the highly visible green fluorescent protein provides a potent tool for the delivery of p53 into cells and the subsequent detection of the protein, both in vitro and in vivo.     

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.     

Ultrafast electron diffraction and direct observation of transient structures in a chemical reaction

Ultrafast electron diffraction is a unique method for the studies of structural changes of complex molecular systems. In this contribution, we report direct ultrafast electron diffraction study of the evolution of short-lived intermediates in the course of a chemical change. Specifically, we observe the transient intermediate in the elimination reaction of 1,2-diiodotetrafluoroethane (C2F4I2) to produce the corresponding ethylene derivative by the breakage of two carbon-iodine, C---I, bonds. The evolution of the ground-state intermediate (C2F4I radical) is directly revealed in the population change of a single chemical bond, namely the second C---I bond. The elimination of two iodine atoms was shown to be nonconcerted, with reaction time of the second C---I bond breakage being 17 +/- 2 ps. The structure of the short-lived C2F4I radical is more favorable to the classical radical structure than to the bridged radical structure. This leap in our ability to record structural changes on the ps and shorter time scales bodes well for many future applications in complex molecular systems.     

Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase

Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.