Intracellular antibodies for proteomics

Visintin, Michela; Meli, Giovanni; Cannistraci, Isabella; Cattaneo, Antonino

doi:10.1016/j.jim.2004.04.014

Cited by 53 publications

(66 citation statements)

References 55 publications

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“…Antibody engineering has allowed the expression of antibody fragments intracellularly 28 and we and other researchers have shown that single variable domains of antibodies are the more efficacious intracellular antibody fragments. 29,30 There are also devised protocols for allow direct selection [31][32][33][34] (and TT & THR, in preparation). Furthermore, we have shown by X-ray crystallography, that single domains do not require intra-domain disulphide bridges to function in cells, providing the basis for single domain libraries with stable domain scaffold for selection against any antigen.…”

Section: Anti-ras Therapeutic Opportunitiesmentioning

confidence: 99%

Interfering with protein-protein interactions: Potential for cancer therapy

Tanaka

Rabbitts²

2008

Cell Cycle

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Genotype-specific cancer therapy promises to engender the era of personalised medicines in which rapid identification of tumour specific gene mutations coupled to rapid methods for efficacious drug identification will be applied. Aberrant signal transduction via protein-protein interactions is generally difficult to target with small molecules. However, macromolecules (macrodrugs) can be developed that interfere with protein-protein interactions by binding with high affinity and specificity to contact surfaces. Inhibitors of mutant RAS and its effector protein interactions affect cancer by attenuating aberrant RAS-dependent signal transduction and would be effective against mutant RAS in dividing cells of overt tumours and in putative cancer stem cells when they move into cell cycle. Results with an antibody fragment blocking effector binding to RAS, illustrates that this is sufficient to prevent cancer. While macrodrugs have inherent problems of bio-distribution and delivery to target cells in patients, their efficacy suggests that efforts to achieve the goal of clinical use should be pursued.

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Section: Anti-ras Therapeutic Opportunitiesmentioning

confidence: 99%

Interfering with protein-protein interactions: Potential for cancer therapy

Tanaka

Rabbitts²

2008

Cell Cycle

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“…The effect of antisense technologies is often not very strong. In a number of cases, even an 80% reduction of mRNA has no effect on the phenotype caused by the protein (3). Furthermore, the interpretation of results obtained from RNAi can be complicated by the up-regulation of other genes, limitation in specificity, and induction of the interferon response (4,5).…”

mentioning

confidence: 99%

Intracellular Kinase Inhibitors Selected from Combinatorial Libraries of Designed Ankyrin Repeat Proteins

Amstutz

Binz

Parizek

et al. 2005

Journal of Biological Chemistry

120

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The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3)-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.

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“…Antibodies are selected directly on the basis of their ability to bind antigen in vivo (60) in a modified version of the two-hybrid method for protein-protein interactions. This method allows the direct screening of yeast cells expressing antibody libraries from different sources (61)(62)(63). These "single-pot libraries of intrabodies" have greatly facilitated the selection of antibody fragments for downstream use as intrabodies in functional studies, providing a user-friendly and robust source of stable antibodies.…”

Section: Using Antibodies As Genes For Protein Silencingmentioning

confidence: 99%

Immunosympathectomy as the first phenotypic knockout with antibodies

Cattaneo

2013

Proc. Natl. Acad. Sci. U.S.A.

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In a PNAS Classic Article published in 1960, Rita Levi-Montalcini offered formal and conclusive proof that endogenous NGF was responsible for the survival of sympathetic neurons in vivo. Thus ended an experimental tour de force lasting a decade, starting with the demonstration that a humoral factor, produced from a tumor transplanted in a chicken embryo, was responsible for stimulating outgrowth of nerve fibers from sympathetic and sensory neurons. From a more general methodological point of view, this work provided a breakthrough in the quest to achieve targeted loss of function and experimentally validate the function of biological molecules. Finally, this work provided an example of the ablation of a specific neuronal subpopulation in an otherwise intact nervous system, an immunological knife of unsurpassed effectiveness and precision. The novelty and the importance of the PNAS Classic Article is discussed here, collocating it within the context of the particular moment of the NGF discovery saga, of Rita Levi-Montalcini's scientific and academic career, and of the general scientific context of those years. This seminal work, involving the use of antibodies for phenotypic knockout in vivo, planted seeds that were to bear new fruit many years later with the advent of monoclonal antibodies and recombinant antibody technologies.anti-NGF antibodies | intrabodies | protein knockout | protein interference

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Intracellular antibodies for proteomics

Cited by 53 publications

References 55 publications

Interfering with protein-protein interactions: Potential for cancer therapy

Interfering with protein-protein interactions: Potential for cancer therapy

Intracellular Kinase Inhibitors Selected from Combinatorial Libraries of Designed Ankyrin Repeat Proteins

Immunosympathectomy as the first phenotypic knockout with antibodies

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