Reduced proliferation and colony formation of head and neck squamous cell carcinoma (HNSCC) after dual targeting of EGFR and hedgehog pathways.

Purpose: The hedgehog signalling pathway (Hh) is frequently active in head and neck squamous cell carcinoma (HNSCC). Overexpressed Hh associates with poor prognosis. The Hh inhibitor vismodegib targets smoothened, and based on molecular data, may prevent resistance to EGFR targeting.

Structure and dynamics of coxsackievirus B4 2A proteinase, an enyzme involved in the etiology of heart disease.

The 2A proteinases (2A(pro)) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A(pro) also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A(pro) (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A(pro) substrate region).

Genetic variability of encephalomyocarditis virus (EMCV) isolates.

In order to evaluate the variability of encephalomyocarditis virus (EMCV), field isolates originating from different European regions and inducing different clinical pictures in pigs have been molecularly characterised. The regions targeted were the poly(C) tract, a part of the 5'-UTR (360 nucleotides), the Leader gene (201 nucleotides), the complete capsid coding region (2502 nucleotides), the 2A gene (403 nucleotides), the end of the 3D polymerase gene (305 nucleotides) and the 3'-UTR (123 nucleotides). Analyses have also been performed on a virulent field isolate, which had been subjected to serial passages in vivo and in vitro resulting, in the case of the in vitro passaged virus, in attenuation, as demonstrated by animal experiments.

Rapid purification of truncated tau proteins: model approach to purification of functionally active fragments of disordered proteins, implication for neurodegenerative diseases.

Truncated tau is of great interest because of its important role in neurofibrillary pathogenesis in Alzheimer's disease (AD). A major obstacle for characterization of detailed biochemical and biological properties of truncated tau species and their fragments has been the lack of reliable and quick purification methods. Uneven distribution of acidic and basic residues in tau determines that the N- and C-terminal tau fragments require entirely different purification conditions.

A thermosensitive mutant of HRV2 2A proteinase: evidence for direct cleavage of eIF4GI and eIF4GII.

Infection of mammalian cells with picornaviruses like entero-, rhino-, and aphthoviruses leads to an inhibition of cap-dependent cellular protein synthesis by the cleavage of both translation initiation factors, eIF4GI and eIF4GII. In entero- and rhinovirus infection this cleavage process is mediated by the viral 2A proteinase (2A(pro)). In order to discriminate between a direct mode of eIF4G cleavage and an indirect cleavage via activation of a cellular proteinase, a thermosensitive 2A(pro) mutant (ts-2A(pro)) of human rhinovirus 2 was employed.

Poly(A)-binding protein interaction with elF4G stimulates picornavirus IRES-dependent translation.

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated, at least in part, by elF4G, which bridges the mRNA termini by simultaneous binding the poly(A)-binding protein (PABP) and the cap-binding protein, elF4E. The poly(A) tail also stimulates translation from the internal ribosome binding sites (IRES) of a number of picornaviruses.

2A proteinase of human rhinovirus cleaves cytokeratin 8 in infected HeLa cells.

Rhino- and enteroviruses encode two proteinases, 2A and 3C, which are responsible for the processing of the viral polyprotein and for cleavage of several cellular proteins. To identify further targets of the 2A proteinase of human rhinovirus serotype 2 (HRV2), an in vitro cleavage assay followed by two-dimensional electrophoresis was employed. Cytokeratin 8, a member of the intermediate filament group of proteins, was found to be proteolytically cleaved in vitro by the 2A proteinase of HRV2 and of coxsackievirus B4 and in vivo during HRV2 infection of HeLa cells.

The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis.

The crystal structure of the 2A proteinase from human rhinovirus serotype 2 (HRV2-2A(pro)) has been solved to 1.95 A resolution. The structure has an unusual, although chymotrypsin-related, fold comprising a unique four-stranded beta sheet as the N-terminal domain and a six-stranded beta barrel as the C-terminal domain.

Rhinovirus 2A proteinase mediated stimulation of rhinovirus RNA translation is additive to the stimulation effected by cellular RNA binding proteins.

The internal ribosome entry site (IRES) of enteroviruses, and especially human rhinoviruses (HRV), functions very inefficiently in rabbit reticulocyte lysates, but can be stimulated by addition of HeLa cell extracts. Two HeLa cell activities have been identified: the A-type activity is due to polypyrimidine tract binding protein and the B-type to unr. In addition HRV and enterovirus IRES function requires a third RNA binding protein, poly(rC) binding protein 2, but this is present in reticulocyte lysates in non-limiting amounts.

Mutational analyses support a model for the HRV2 2A proteinase.

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular ("cis") and intermolecular ("trans") activities.

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.

Human rhinovirus 2A proteinase mutant and its second-site revertants.

The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130-->Tyr almost completely inhibited enzyme activity at 37 degrees C; activity was, however, partially restored by the following exchanges: Ser-27-->Pro, His-135-->Arg or His-137-->Arg.

Proteolytically active 2A proteinase of human rhinovirus 2 is toxic for Saccharomyces cerevisiae but does not cleave the homologues of eIF-4 gamma in vivo or in vitro.

During the replication of rhino- and enteroviruses, the translation initiation factor elF-4 gamma is specifically cleaved by the virally encoded 2 A proteinase. This cleavage has been proposed to lead to the inability of the host cell to translate its own capped mRNA and to stimulate internal initiation of protein synthesis from the viral mRNA. However, a direct causal relationship between these effects and 2A proteinase-mediated cleavage of elF-4 gamma has remained difficult to prove, mainly because of the toxicity of the 2A proteinase in mammalian expression systems.

Picornavirus 2A proteinase-mediated stimulation of internal initiation of translation is dependent on enzymatic activity and the cleavage products of cellular proteins.

Poliovirus and human rhinovirus 2A proteinases are known to stimulate translation initiation on the cognate viral Internal Ribosome Entry Segments (IRESes). The molecular mechanism of this translational transactivation was investigated in vitro using dicistronic mRNAs containing picornaviral IRESes as the intercistronic spacer and purified human rhinovirus type 2 and coxsackievirus B4 2A proteinases. The stimulation achieved on the HRV2 IRES in the presence of the cognate 2A proteinase at 1 microgram/ml was twofold; the maximum stimulation at 100 micrograms/ml was fivefold.

Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma.

Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.

2A proteinases of coxsackie- and rhinovirus cleave peptides derived from eIF-4 gamma via a common recognition motif.

The cleavage specificities of the 2A proteinases from coxsackievirus B4 (CVB4) and human rhinovirus 2 (HRV2) on oligopeptide substrates have been determined. Comparison of the specificity of CVB4 2A proteinase with that of HRV2 2A proteinase allowed cleavable peptides to be designed using the common motif IIe/Leu-X-Thr-X*Gly; little resemblance to the viral cleavage site remained. The data also allowed the prediction of three possible cleavage sites for 2A proteinases on eIF-4 gamma; two peptides derived from these sequences were cleaved by both 2A proteinases.

Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus.

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4 gamma. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4 gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease.

Purification of two picornaviral 2A proteinases: interaction with eIF-4 gamma and influence on in vitro translation.

A mammalian cell infected with a human rhinovirus or enterovirus has a much reduced capability to translate capped mRNAs (the host cell shutoff), while still allowing translation of uncapped viral RNA. Biochemical and genetic evidence suggests that the viral proteinase 2A induces cleavage of the eukaryotic initiation factor (eIF) 4 gamma (also known as p220) component of eIF-4 (formerly called eIF-4F). However, neither the mechanism underlying the specific proteolysis of eIF-4 gamma nor the influence of this cleavage on the translation of capped mRNAs has been clarified.

Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A.

Proteinase 2A of human rhinovirus serotype 2 (HRV2 2A) was expressed in Escherichia coli and partially purified; the preparation was used to study various enzymatic parameters. Using a 16-amino acid peptide representing the native cleavage region of HRV2 2A, an apparent Km value of 5.4 x 10(-4) mol/liter was determined.

Using proteinase trapping to detect revertants of inactive rhinoviral 2A proteinase mutants.

The 2A proteinase of human rhinovirus 2 cleaves itself off the growing polyprotein at its own N terminus during translation; this property was used to develop an in vivo screening system with the lacZ gene fragment of M13mp18. The fusion of an active 2A proteinase to the C-terminus of the alpha-fragment did not affect alpha-complementation, as the proteinase cleaved itself off the alpha-fragment. However, an inactive 2A proteinase remained fused to the alpha-fragment hindering alpha-complementation.

In vitro inhibition of cytochrome P-450 reductases from pig liver microsomes by garlic extracts.

The activity of microsomal NADPH-cytochrome-P-450-reductase and NADH-cytochrome-b5-reductase are inhibited after the addition of an aqueous extract of a pharmaceutical preparation of garlic (Allium sativum, L.) to buffer-suspended microsomes. Incubation of garlic extract with isolated pig liver microsomes also decreases the activity of cytochrome P-450-dependent ethoxycoumarin deethylation.

Proteinase trapping: screening for viral proteinase mutants by alpha complementation.

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment.

Substrate requirements of a human rhinoviral 2A proteinase.

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2.

Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites.

Twenty-nine early promoters from bacteriophage T4 and 14 early promoters from bacteriophage T6 were isolated using vector M13HDL17, a promoterless derivative of M13mp8 carrying a linker sequence, the bacteriophage lambda-terminator tR1, and the lacZ' gene including part of its ribosome-binding site. The consensus sequence for the T4 promoters is: (sequence; see text). Ribosome-binding sites of T4 share the sequence: 5'.