RESUMO
Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed.
Assuntos
Terapia Genética/métodos , Proteínas Nucleares , Proteínas/genética , Transdução Genética , Proteína do Homeodomínio de Antennapedia , Produtos do Gene tat/genética , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/genética , Proteínas Estruturais Virais/genéticaRESUMO
The dNTpase enzyme has previously been shown to specifically hydrolyse monodeoxyribonucleoside triphosphates (dNTPs). The remnant nucleotide resulting from this hydrolysis lacks the terminal phosphate and is covalently attached as part of a 3 kDa species, which we have termed the product nucleotide binding particle or "PNBP." PNBP is resistant to numerous nucleases and RNases, suggesting that it is not a nucleic acid polymer. Given that the exclusive specificity of dNTPase for dNTPs suggests some associative cellular role for the enzyme in polynucleotide maintenance, the interaction of dNTPase with various nucleic acids has now been examined. It is demonstrated that dNTPase activity is significantly inhibited by addition of single-stranded DNA or tRNA, but not rRNA. The data presented also suggest that thio-dATP can substitute for conventional phosphoester dATP in the enzymatic reaction. It is also demonstrated that the dNTPase enzyme comprises both heat/proteolysis/denaturant stable and heat/proteolysis/denaturant-sensitive components and we propose that this stable component may be the precursor to liganded PNBP.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , DNA/farmacologia , Temperatura Alta , RNA/farmacologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Animais , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/farmacologia , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxiadenina/farmacologia , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Fígado/enzimologia , Peso Molecular , Desnaturação Proteica , RNA/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico/farmacologia , RNA de Transferência/metabolismo , RNA de Transferência/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Suínos , LevedurasRESUMO
A three zinc-finger protein that binds specifically to the cDNA representing the unique fusion gene BCR:Abl, associated with acute lymphoblastic leukaemia, has previously been characterised. At this breakpoint, a sequence homology of 8/9 bp exists between the BCR:Abl (fusion) and c-ABL: (parental) target sequences. We show that the three zinc-finger protein discriminates poorly between the fusion (BCR:Abl) and parental (ABL:) sequence (K:(d)s of 42.8 and 65.1 nM, respectively). In order to improve the discriminatory properties of this protein, and to demonstrate the utility of current zinc-finger databases, we have added a fourth zinc-finger to the original three zinc-finger protein. This fourth finger recognises a 3 bp subsite derived from the BCR: portion of the breakpoint and is not present in c-ABL: This novel four finger protein, which now recognises a 12 bp sequence, demonstrates improved specific binding to BcrAbl (K:(d )= 17 nM). More significantly we have shown that there is now enhanced discrimination between BcrAbl and ABL: sequences by the four finger protein than the original three finger protein.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Fusão bcr-abl/genética , Oncogenes/genética , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , DNA/genética , Proteínas de Ligação a DNA/genética , Genes abl/genética , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcr , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Protein transduction can be described as the direct uptake by the cell of exogenous proteins/peptides or protein/peptide:chemical complexes, as a result of a specific property of the protein/peptide component. In this review, the three most widely studied protein transducing activities are described, with particular emphasis on the TAT protein transduction domain. Current progress in protein transduction technology suggests the potential development of a variety of molecular and cell biology tools that will enable researchers to by-pass conventional genetic routes for modulating the cells' biological activity, thus negating many of the problems associated with genetic intervention. The potential application of this class of molecule in the development of tools for the study of senescent populations is discussed.
Assuntos
Proteínas/genética , Transdução Genética , Animais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/farmacologia , Divisão Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Humanos , Fragmentos de Peptídeos/farmacologia , Transdução Genética/métodosRESUMO
The 'malic' enzyme (ME) gene promoter contains three main regulatory regions. One of these, the direct repeat element (DRE), contains tandem degenerate Sp1-binding sites separated by a 3 bp intervening sequence. We now show that a previously unreported 95 kDa protein, which we have designated DRP-1, binds strongly to the DRE region in a highly specific manner. Western-blot analysis confirms that this protein is not Sp1, which has been shown to bind to similar degenerate sites. Competitive binding assays using purified DRP-1 further reveal that neither non-specific nor Sp1-consensus-site-containing oligonucleotides can displace those complexes formed between DRP-1 and the DRE sequence, thus confirming sequence-specific binding by this protein. SDS/PAGE analysis of DRE-protein complexes isolated by direct excision and transplantation from retardation gels confirms the presence of the 95 kDa protein and, in addition, suggests that more than one binding site exists for this protein within the DRE. This is in accord with the repeated nature of the DRE DNA sequence which contains two CACC box motifs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Malato Desidrogenase/genética , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , DNA/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Sensibilidade e Especificidade , Fator de Transcrição Sp1/metabolismoRESUMO
The interactions of several porphyrins with a 74 base-pair DNA sequence have been examined by footprinting and chemical protection methods. Tetra-(4-N-methyl-(pyridyl)) porphyrin (TMPy), two of its metal complexes and tetra-(4-trimethylanilinium) porphyrin (TMAP) bind to closely similar AT-rich sequences. The three TMPy ligands produce modest changes in DNA structure and base accessibility on binding, in contrast to the large-scale conformational changes observed with TMAP. Molecular modelling studies have been performed on TMPy and TMAP bound in the AT-rich minor groove of an oligonucleotide. These have shown that significant structural change is needed to accommodate the bulky trimethyl substituent groups of TMAP, in contrast to the facile minor groove fit of TMPy.
Assuntos
DNA/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Porfirinas/química , Sequência de Bases , Sítios de Ligação , DNA/efeitos dos fármacos , Pegada de DNA , Modelos Moleculares , Dados de Sequência Molecular , Porfirinas/farmacologiaAssuntos
Escherichia coli/genética , Glutationa Transferase/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/isolamento & purificação , Fator de Transcrição TFIID , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Arabidopsis thaliana protoplasts, isolated from a cell suspension, regenerated cell walls and then exhibited sustained cell divisions. Within 10 days after isolation as many as 40% of the protoplast-derived cells divided. Protoplast-derived calli exhibited a wide range of developmental responses, including apparent embryogenesis. The cell suspension has been cryopreserved.
RESUMO
The molecular structure of the DNA-intercalating ligand tetra-(4-N-methylpyridyl) porphin has been determined by X-ray crystallography. The porphyrin has a precise centre of symmetry; the central core is planar, with the N-methylpyridyl groups inclined to it at angles of 66-72 degrees. Molecular modelling of this structure into TpA and CpG sites of intercalated DNA, has been performed, and approximate energetics calculated. It has been shown that only the CpG site can have full ligand intercalation, since the thymine methyl group sterically hinders such geometry at TpA sites. Modelling indicates the importance of electrostatic effects in the low-energy forms of intercalated and part-intercalated complexes at both sequences.
