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Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy.Annual Review of Pharmacology and... 2015Designed ankyrin repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their... (Review)
Review
Designed ankyrin repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their robustness and extreme stability, they allow a multitude of more advanced formats and applications. This review highlights recent advances in DARPin design, illustrates their properties, and gives some examples of their use. In research, they have been established as intracellular, real-time sensors of protein conformations and as crystallization chaperones. For future therapies, DARPins have been developed by advanced, structure-based protein engineering to selectively induce apoptosis in tumors by uncoupling surface receptors from their signaling cascades. They have also been used successfully for retargeting viruses. In ongoing clinical trials, DARPins have shown good safety and efficacy in macular degeneration diseases. These developments all ultimately exploit the high stability, solubility, and aggregation resistance of these molecules, permitting a wide range of conjugates and fusions to be produced and purified.
Topics: Angiogenesis Inhibitors; Animals; Ankyrin Repeat; Drug Carriers; Drug Design; Genetic Therapy; Humans; Models, Molecular; Peptide Library; Protein Conformation; Protein Engineering; Recombinant Proteins; Structure-Activity Relationship
PubMed: 25562645
DOI: 10.1146/annurev-pharmtox-010611-134654 -
Viruses Oct 2022Designed ankyrin repeat proteins (DARPins) are engineered proteins comprising consensus designed ankyrin repeats as scaffold. Tightly packed repeats form a continuous... (Review)
Review
Designed ankyrin repeat proteins (DARPins) are engineered proteins comprising consensus designed ankyrin repeats as scaffold. Tightly packed repeats form a continuous hydrophobic core and a large groove-like solvent-accessible surface that creates a binding surface. DARPin domains recognizing a target of interest with high specificity and affinity can be generated using a synthetic combinatorial library and in vitro selection methods. They can be linked together in a single molecule to build multispecific and multifunctional proteins without affecting expression or function. The modular architecture of DARPins offers unprecedented possibilities of design and opens avenues for innovative antiviral strategies.
Topics: Designed Ankyrin Repeat Proteins; Virus Internalization; Ankyrin Repeat; Proteins; HIV Fusion Inhibitors; Solvents
PubMed: 36298797
DOI: 10.3390/v14102242 -
International Journal of Molecular... Jun 2017The ankyrin repeat domain 1 (ANKRD1) protein is a cardiac-specific stress-response protein that is part of the muscle ankyrin repeat protein family. ANKRD1 is... (Review)
Review
The ankyrin repeat domain 1 (ANKRD1) protein is a cardiac-specific stress-response protein that is part of the muscle ankyrin repeat protein family. ANKRD1 is functionally pleiotropic, playing pivotal roles in transcriptional regulation, sarcomere assembly and mechano-sensing in the heart. Importantly, cardiac ANKRD1 has been shown to be highly induced in various cardiomyopathies and in heart failure, although it is still unclear what impact this may have on the pathophysiology of heart failure. This review aims at highlighting the known properties, functions and regulation of ANKRD1, with focus on the underlying mechanisms that may be involved. The current views on the actions of ANKRD1 in cardiovascular disease and its utility as a candidate cardiac biomarker with diagnostic and/or prognostic potential are also discussed. More studies of ANKRD1 are warranted to obtain deeper functional insights into this molecule to allow assessment of its potential clinical applications as a diagnostic or prognostic marker and/or as a possible therapeutic target.
Topics: Animals; Ankyrin Repeat; Biomarkers; Carrier Proteins; Disease Susceptibility; Gene Expression Regulation; Heart Diseases; Humans; Muscle Proteins; Myocytes, Cardiac; Nuclear Proteins; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Proteolysis; Repressor Proteins
PubMed: 28672880
DOI: 10.3390/ijms18071362 -
International Journal of Molecular... Jan 2021Ankyrin repeat (AR) domains are considered the most abundant repeat motif found in eukaryotic proteins. AR domains are predominantly known to mediate specific... (Review)
Review
Ankyrin repeat (AR) domains are considered the most abundant repeat motif found in eukaryotic proteins. AR domains are predominantly known to mediate specific protein-protein interactions (PPIs) without necessarily recognizing specific primary sequences, nor requiring strict conformity within its own primary sequence. This promiscuity allows for one AR domain to recognize and bind to a variety of intracellular substrates, suggesting that AR-containing proteins may be involved in a wide array of functions. Many AR-containing proteins serve a critical role in biological processes including the ubiquitylation signaling pathway (USP). There is also strong evidence that AR-containing protein malfunction are associated with several neurological diseases and disorders. In this review, the structure and mechanism of key AR-containing proteins are discussed to suggest and/or identify how each protein utilizes their AR domains to support ubiquitylation and the cascading pathways that follow upon substrate modification.
Topics: Animals; Ankyrin Repeat; Carcinogenesis; Endopeptidases; Humans; Models, Molecular; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Signal Transduction; Ubiquitin; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 33435370
DOI: 10.3390/ijms22020609 -
Biochemistry Dec 2006Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein... (Review)
Review
Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.
Topics: Amino Acid Motifs; Animals; Ankyrin Repeat; Humans; Models, Molecular; Protein Interaction Domains and Motifs; Protein Interaction Mapping
PubMed: 17176038
DOI: 10.1021/bi062188q -
Anti-inflammatory & Anti-allergy Agents... 2017Antibodies constitute an important drug development platform for drugs to treat several ophthalmic, oncologic, and immunologic conditions, but due to limitations... (Review)
Review
BACKGROUND
Antibodies constitute an important drug development platform for drugs to treat several ophthalmic, oncologic, and immunologic conditions, but due to limitations inherent in antibody production and structure, a wide range of other protein binding scaffolds are being investigated. Designed ankyrin repeat proteins (DARPins) are simple to produce and offer a range of advantages over antibodies because of their stability, high binding affinity, and rigid structure.
OBJECTIVE
DARPins are being developed for a wide variety of medical applications, and the most studied molecule, abicipar pegol, is used to treat chorioretinal vascular diseases. This mini-review will discuss the current state of DARPin technology and will summarize drug development with a focus on abicipar.
METHODS
PubMed searches with keywords "DARPin" and "designed ankyrin repeat proteins" were performed and the reference lists of identified articles were examined for additional research material. Studies using DARPin molecules were identified at Clinicaltrials. gov. Non-peer reviewed data were found through Google searches of pertinent websites.
RESULTS
Abicipar prevents angiogenesis by binding all isoforms of vascular endothelial growth factor (VEGF)-A with single-digit picomolar affinity. Abicipar has a long intraocular half-life in rabbits and has produced promising results in pre-clinical studies. Pivotal phase III registration trials for the treatment of neovascular age-related macular degeneration are ongoing and a phase II/III trial for the treatment of diabetic macular edema has been announced.
CONCLUSION
Abicipar pegol has the potential to effectively treat chorioretinal vascular conditions with an extended duration of action beyond those of currently used anti-VEGF drugs.
Topics: Angiogenesis Inhibitors; Animals; Ankyrin Repeat; Choroid Diseases; Humans; Retinal Diseases; Vascular Diseases
PubMed: 28464780
DOI: 10.2174/1871523016666170502115816 -
Protein Science : a Publication of the... Jun 2004The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of... (Review)
Review
The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.
Topics: Amino Acid Sequence; Ankyrin Repeat; Computer Simulation; Molecular Sequence Data; Protein Binding; Proteins
PubMed: 15152081
DOI: 10.1110/ps.03554604 -
Nature Cell Biology Oct 2019Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from...
Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.
Topics: Animals; Ankyrin Repeat; Cell Membrane; Cells, Cultured; Cytoskeletal Proteins; HEK293 Cells; HeLa Cells; Humans; Mice; Models, Molecular; Morphogenesis; Neurons; Protein Domains; Rats; Transcription Factors
PubMed: 31548610
DOI: 10.1038/s41556-019-0381-7 -
Protein Science : a Publication of the... Aug 2023Numerous age-linked diseases are rooted in protein misfolding; this has motivated the development of small molecules and therapeutic antibodies that target the...
Numerous age-linked diseases are rooted in protein misfolding; this has motivated the development of small molecules and therapeutic antibodies that target the aggregation of disease-linked proteins. Here we explore another approach: molecular chaperones with engineerable protein scaffolds such as the ankyrin repeat domain (ARD). We tested the ability of cpSRP43, a small, robust, ATP- and cofactor-independent plant chaperone built from an ARD, to antagonize disease-linked protein aggregation. cpSRP43 delays the aggregation of multiple proteins including the amyloid beta peptide (Aβ) associated with Alzheimer's disease and α-synuclein associated with Parkinson's disease. Kinetic modeling and biochemical analyses show that cpSRP43 targets early oligomers during Aβ aggregation, preventing their transition to a self-propagating nucleus on the fibril surface. Accordingly, cpSRP43 rescued neuronal cells from the toxicity of extracellular Aβ42 aggregates. The substrate-binding domain of cpSRP43, composed primarily of the ARD, is necessary and sufficient to prevent Aβ42 aggregation and protect cells against Aβ42 toxicity. This work provides an example in which an ARD chaperone non-native to mammalian cells harbors anti-amyloidal activity, which may be exploited for bioengineering.
Topics: Animals; Amyloid beta-Peptides; Ankyrin Repeat; Molecular Chaperones; Alzheimer Disease; Parkinson Disease; Amyloid; Mammals
PubMed: 37433015
DOI: 10.1002/pro.4728 -
Plant Cell Reports Feb 2021The ankyrin repeat-containing protein MdANK2B was identified to contribute to increasing resistance to salt stress and decreasing sensitivity to ABA in Malus domestica....
The ankyrin repeat-containing protein MdANK2B was identified to contribute to increasing resistance to salt stress and decreasing sensitivity to ABA in Malus domestica. Ankyrin (ANK) repeat-containing proteins occur widely in prokaryotes, eukaryotes, and even in some viruses and play a critical role in plant growth and development, as well as the response to biotic and abiotic stress. However, the function of ANK repeat-containing proteins in apple (Malus domestica) has not yet been investigated. Here, we identified apple MdANK2B based on homology analysis with the Arabidopsis ANK repeat-containing proteins AtAKR2A and AtAKR2B. MdANK2B was found to be localized in the cytoplasm, and its encoding gene was highly expressed in both apple leaves and fruits. In addition, MdANK2B gene expression was highly induced by salt stresses and abscisic acid (ABA). Overexpression of MdANK2B increased resistance to salt stress and decreased sensitivity to ABA in both transgenic apple calli and seedlings. In addition, overexpression of MdANK2B reduced the accumulation of reactive oxygen species (ROS) by enhancing the activity of antioxidant enzymes in response to salt stress. Our data revealed the role of MdANK2B in response to salt stress and ABA treatment in apple, which widens the known functions of ANK repeat-containing proteins in response to abiotic stress.
Topics: Abscisic Acid; Ankyrin Repeat; Arabidopsis Proteins; Gene Expression; Malus; Molecular Chaperones; Plant Growth Regulators; Plant Proteins; Reactive Oxygen Species; Salt Stress; Salt Tolerance; Stress, Physiological
PubMed: 33331953
DOI: 10.1007/s00299-020-02642-9