Intrabody (protein)

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In molecular biology, an intrabody (from intracellular and antibody ) is an antibody that works within the cell to bind to an intracellular protein. [1] Due to the lack of a reliable mechanism for bringing antibodies into a living cell from the extracellular environment, this typically requires the expression of the antibody within the target cell, which can be accomplished in transgenic animals [2] or by gene therapy. As a result, intrabodies are defined as antibodies that have been modified for intracellular localization, and the term has rapidly come to be used even when antibodies are produced in prokaryotes or other non-target cells. [3] This term can apply to several types of protein targeting: the antibody may remain in the cytoplasm, or it may have a nuclear localization signal, [4] or it may undergo cotranslational translocation across the membrane into the lumen of the endoplasmic reticulum, provided that it is retained in that compartment through a KDEL sequence. [5]

Because naturally occurring antibodies are optimised to be secreted from the cell, cytosolic intrabodies require special alterations, including the use of single-chain antibodies (scFvs), modification of immunoglobulin VL domains for hyperstability, [6] selection of antibodies resistant to the more reducing cytosolic environment, [7] or expression as a fusion protein with maltose binding protein or other stable intracellular proteins. [8] Such optimizations have improved the stability and structure of intrabodies, allowing the publication of a variety of promising applications against hepatitis B, [9] avian influenza, [10] prion diseases, [11] inflammation, [12] Parkinson's disease, [13] and Huntington's disease. [14] Optimizations required for cytosolic intrabodies are not needed for ER retained intrabodies, which fold in the compartment in which antibodies are naturally produced. Since the 1990s ER intrabodies have been used in various research areas to knock down membrane proteins and secreted proteins. [15]


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<span class="mw-page-title-main">Prion</span> Pathogenic type of misfolded protein

A prion is a misfolded protein that can induce misfolding of normal variants of the same protein and trigger cellular death. Prions cause prion diseases known as transmissible spongiform encephalopathies (TSEs) that are transmissible, fatal neurodegenerative diseases in humans and animals. The proteins may misfold sporadically, due to genetic mutations, or by exposure to an already misfolded protein. The consequent abnormal three-dimensional structure confers on them the ability to cause misfolding of other proteins.

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Inositol trisphosphate or inositol 1,4,5-trisphosphate abbreviated InsP3 or Ins3P or IP3 is an inositol phosphate signaling molecule. It is made by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, by phospholipase C (PLC). Together with diacylglycerol (DAG), IP3 is a second messenger molecule used in signal transduction in biological cells. While DAG stays inside the membrane, IP3 is soluble and diffuses through the cell, where it binds to its receptor, which is a calcium channel located in the endoplasmic reticulum. When IP3 binds its receptor, calcium is released into the cytosol, thereby activating various calcium regulated intracellular signals.

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<span class="mw-page-title-main">Amyloid</span> Insoluble protein aggregate with a fibrillar morphology

Amyloids are aggregates of proteins characterised by a fibrillar morphology of typically 7–13 nm in diameter, a β-sheet secondary structure and ability to be stained by particular dyes, such as Congo red. In the human body, amyloids have been linked to the development of various diseases. Pathogenic amyloids form when previously healthy proteins lose their normal structure and physiological functions (misfolding) and form fibrous deposits within and around cells. These protein misfolding and deposition processes disrupt the healthy function of tissues and organs.

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<span class="mw-page-title-main">Amyloid beta</span> Group of peptides

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ATP7A, also known as Menkes' protein (MNK), is a copper-transporting P-type ATPase which uses the energy arising from ATP hydrolysis to transport Cu(I) across cell membranes. The ATP7A protein is a transmembrane protein and is expressed in the intestine and all tissues except liver. In the intestine, ATP7A regulates Cu(I) absorption in the human body by transporting Cu(I) from the small intestine into the blood. In other tissues, ATP7A shuttles between the Golgi apparatus and the cell membrane to maintain proper Cu(I) concentrations in the cell and provides certain enzymes with Cu(I). The X-linked, inherited, lethal genetic disorder of the ATP7A gene causes Menkes disease, a copper deficiency resulting in early childhood death.

<span class="mw-page-title-main">Tissue transglutaminase</span> Protein-coding gene in the species Homo sapiens

Tissue transglutaminase is a 78-kDa, calcium-dependent enzyme of the protein-glutamine γ-glutamyltransferases family. Like other transglutaminases, it crosslinks proteins between an ε-amino group of a lysine residue and a γ-carboxamide group of glutamine residue, creating an inter- or intramolecular bond that is highly resistant to proteolysis. Aside from its crosslinking function, tTG catalyzes other types of reactions including deamidation, GTP-binding/hydrolyzing, and isopeptidase activities. Unlike other members of the transglutaminase family, tTG can be found both in the intracellular and the extracellular spaces of various types of tissues and is found in many different organs including the heart, the liver, and the small intestine. Intracellular tTG is abundant in the cytosol but smaller amounts can also be found in the nucleus and the mitochondria. Intracellular tTG is thought to play an important role in apoptosis. In the extracellular space, tTG binds to proteins of the extracellular matrix (ECM), binding particularly tightly to fibronectin. Extracellular tTG has been linked to cell adhesion, ECM stabilization, wound healing, receptor signaling, cellular proliferation, and cellular motility.

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<span class="mw-page-title-main">Proteinopathy</span> Medical condition

In medicine, proteinopathy, or proteopathy, protein conformational disorder, or protein misfolding disease, is a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way or they can lose their normal function. The proteinopathies include such diseases as Creutzfeldt–Jakob disease and other prion diseases, Alzheimer's disease, Parkinson's disease, amyloidosis, multiple system atrophy, and a wide range of other disorders. The term proteopathy was first proposed in 2000 by Lary Walker and Harry LeVine.

<span class="mw-page-title-main">PLA2G6</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">Neurofilament light polypeptide</span> Protein-coding gene in the species Homo sapiens

Neurofilament light polypeptide, also known as neurofilament light chain, abbreviated to NF-L or Nfl and with the HGNC name NEFL is a member of the intermediate filament protein family. This protein family consists of over 50 human proteins divided into 5 major classes, the Class I and II keratins, Class III vimentin, GFAP, desmin and the others, the Class IV neurofilaments and the Class V nuclear lamins. There are four major neurofilament subunits, NF-L, NF-M, NF-H and α-internexin. These form heteropolymers which assemble to produce 10nm neurofilaments which are only expressed in neurons where they are major structural proteins, particularly concentrated in large projection axons. Axons are particularly sensitive to mechanical and metabolic compromise and as a result axonal degeneration is a significant problem in many neurological disorders. The detection of neurofilament subunits in CSF and blood has therefore become widely used as a biomarker of ongoing axonal compromise. The NF-L protein is encoded by the NEFL gene. Neurofilament light chain is a biomarker that can be measured with immunoassays in cerebrospinal fluid and plasma and reflects axonal damage in a wide variety of neurological disorders. It is a useful marker for disease monitoring in amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, and more recently Huntington's disease. It is also promising marker for follow-up of patients with brain tumors. Higher levels of blood or CSF NF-L have been associated with increased mortality, as would be expected as release of this protein reflects ongoing axonal loss. Recent work performed as a collaboration between EnCor Biotechnology Inc. and the University of Florida showed that the NF-L antibodies employed in the most widely used NF-L assays are specific for cleaved forms of NF-L generated by proteolysis induced by cell death. Methods used in different studies for NfL measurement are sandwich enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence, and high-sensitive single molecule array (SIMOA).

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<span class="mw-page-title-main">JUNQ and IPOD</span> Types of cytosolic protein inclusion bodies

JUNQ and IPOD are types of cytosolic protein inclusion bodies in eukaryotes.

<span class="mw-page-title-main">Stimulator of interferon genes</span> Protein-coding gene in the species Homo sapiens

Stimulator of interferon genes (STING), also known as transmembrane protein 173 (TMEM173) and MPYS/MITA/ERIS is a protein that in humans is encoded by the STING1 gene.

Stefan Dübel is a German biologist. Since October 2002, he has been a full professor at the University of Braunschweig and head of the Biotechnology Department of the Institute of Biochemistry, Biotechnology and Bioinformatics. His work is centred around protein engineering, phage display and recombinant antibodies.

References

  1. ↑ Chen, SY; Bagley, J; Marasco, WA (1994). "Intracellular antibodies as a new class of therapeutic molecules for gene therapy". Human Gene Therapy. 5 (5): 595–601. doi:10.1089/hum.1994.5.5-595. PMID   7914435.
  2. ↑ Marschall, AL; Single, FN; Schlarmann, K; Bosio, A; Strebe, N; van den Heuvel, J; Frenzel, A; Dübel, S (2014). "Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies". mAbs. 6 (6): 1394–401. doi: 10.4161/mabs.34377 . PMC   4622715 . PMID   25484057.
  3. ↑ Cohen, PA; Mani, JC; Lane, DP (1998). "Characterization of a new intrabody directed against the N-terminal region of human p53". Oncogene. 17 (19): 2445–56. doi:10.1038/sj.onc.1202190. PMID   9824155.
  4. ↑ Mhashilkar, AM; Bagley, J; Chen, SY; Szilvay, AM; Helland, DG; Marasco, WA (1995). "Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies". The EMBO Journal. 14 (7): 1542–51. doi:10.1002/j.1460-2075.1995.tb07140.x. PMC   398241 . PMID   7537216.
  5. ↑ Yurong Yang Wheeler; Timothy E. Kute; Mark C. Willingham; Si-Yi Chen; David C. Sane (2003). "Intrabody-based strategies for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis". The FASEB Journal. 17 (12): 1733–5. doi: 10.1096/fj.02-0942fje . PMID   12958192.
  6. ↑ Cohen, PA; Mani, JC; Lane, DP (1998). "Characterization of a new intrabody directed against the N-terminal region of human p53". Oncogene. 17 (19): 2445–56. doi:10.1038/sj.onc.1202190. PMID   9824155.
  7. ↑ Auf Der Maur, A; Escher, D; Barberis, A (2001). "Antigen-independent selection of stable intracellular single-chain antibodies". FEBS Letters. 508 (3): 407–12. doi:10.1016/S0014-5793(01)03101-5. PMID   11728462.
  8. ↑ Shaki-Loewenstein, S; Zfania, R; Hyland, S; Wels, WS; Benhar, I (2005). "A universal strategy for stable intracellular antibodies". Journal of Immunological Methods. 303 (1–2): 19–39. doi:10.1016/j.jim.2005.05.004. PMID   16045924.
  9. ↑ Serruys, B; Van Houtte, F; Verbrugghe, P; Leroux-Roels, G; Vanlandschoot, P (2009). "Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice". Hepatology. 49 (1): 39–49. doi:10.1002/hep.22609. PMID   19085971.
  10. ↑ Mukhtar, MM; Li, S; Li, W; Wan, T; Mu, Y; Wei, W; Kang, L; Rasool, ST; et al. (2009). "Single-chain intracellular antibodies inhibit influenza virus replication by disrupting interaction of proteins involved in viral replication and transcription". The International Journal of Biochemistry & Cell Biology. 41 (3): 554–60. doi:10.1016/j.biocel.2008.07.001. PMID   18687409.
  11. ↑ Filesi, I; Cardinale, A; Mattei, S; Biocca, S (2007). "Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrP(Sc) formation". Journal of Neurochemistry. 101 (6): 1516–26. doi:10.1111/j.1471-4159.2006.04439.x. PMID   17542810.
  12. ↑ Strebe, N; Guse, A; Schüngel, M; Schirrmann, T; Hafner, M; Jostock, T; Hust, M; Müller, W; Dübel, S (2009). "Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies". Journal of Immunological Methods. 341 (1–2): 30–40. doi:10.1016/j.jim.2008.10.012. PMID   19038261.
  13. ↑ Zhou, C; Przedborski, S (2009). "Intrabody and Parkinson's disease". Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1792 (7): 634–42. doi:10.1016/j.bbadis.2008.09.001. PMC   2745095 . PMID   18834937.
  14. ↑ Cardinale, A; Biocca, S (2008). "The potential of intracellular antibodies for therapeutic targeting of protein-misfolding diseases". Trends in Molecular Medicine. 14 (9): 373–80. doi:10.1016/j.molmed.2008.07.004. PMID   18693139.
  15. ↑ Marschall, AL; Dübel, S; Böldicke, T (2015). "Specific in vivo knockdown of protein function by intrabodies". mAbs. 7 (6): 1010–35. doi: 10.1080/19420862.2015.1076601 . PMC   4966517 . PMID   26252565.
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