-
FASEB Journal : Official Publication of... Apr 2021Cardiac ankyrin repeat protein (CARP) is a cardiac-specific stress-response protein which exerts diverse effects to modulate cardiac remodeling in response to...
Cardiac ankyrin repeat protein (CARP) is a cardiac-specific stress-response protein which exerts diverse effects to modulate cardiac remodeling in response to pathological stimuli. We examined the role of CARP in postnatal cardiac development and function under basal conditions in mice. Transgenic mice that selectively overexpressed CARP in heart (CARP Tg) exhibited dilated cardiac chambers, impaired heart function, and cardiac fibrosis as assessed by echocardiography and histological staining. Furthermore, the mice had a shorter lifespan and reduced survival rate in response to ischemic acute myocardial infarction. Immunofluorescence demonstrated the overexpressed CARP protein was predominantly accumulated in the nuclei of cardiomyocytes. Microarray analysis revealed that the nuclear localization of CARP was associated with the suppression of calcium-handling proteins. In vitro experiments revealed that CARP overexpression resulted in decreased cell contraction and calcium transient. In post-mortem cardiac specimens from patients with dilated cardiomyopathy and end-stage heart failure, CARP was significantly increased. Taken together, our data identified CARP as a crucial contributor in dilated cardiomyopathy and heart failure which was associated with its regulation of calcium-handling proteins.
Topics: Animals; Cardiomyopathy, Dilated; Heart Failure; Humans; Mice; Muscle Proteins; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Nuclear Proteins; Repressor Proteins
PubMed: 33734499
DOI: 10.1096/fj.201902802RR -
International Journal of Molecular... Aug 2023Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate... (Review)
Review
Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate protein-protein interactions and a KH domain that mediates RNA or single-stranded DNA binding. ANKHD1 interacts with proteins in several crucial signalling pathways, including receptor tyrosine kinase, JAK/STAT, mechanosensitive Hippo (YAP/TAZ), and p21. Studies into the role of ANKHD1 in cancer cell lines demonstrate a crucial role in driving uncontrolled cellular proliferation and growth, enhanced tumorigenicity, cell cycle progression through the S phase, and increased epithelial-to-mesenchymal transition. Furthermore, at a clinical level, the increased expression of ANKHD1 has been associated with greater tumour infiltration, increased metastasis, and larger tumours. Elevated ANKHD1 resulted in poorer prognosis, more aggressive growth, and a decrease in patient survival in numerous cancer types. This review aims to gather the current knowledge about ANKHD1 and explore its molecular properties and functions, focusing on the protein's role in cancer at both a cellular and clinical level.
Topics: Humans; Neoplasms; Hyperplasia; Aggression; Ankyrin Repeat; Cell Division; RNA-Binding Proteins
PubMed: 37629022
DOI: 10.3390/ijms241612834 -
Nucleic Acid Therapeutics Jun 2018Although recent clinical successes of antisense, splice-switching, and siRNA oligonucleotides have established the therapeutic utility of this novel class of medicines,... (Review)
Review
Although recent clinical successes of antisense, splice-switching, and siRNA oligonucleotides have established the therapeutic utility of this novel class of medicines, the efficient systemic application for non-liver targets remains elusive. Exploitation of active receptor-mediated targeting followed by efficient and productive cellular uptake is required for enabling the therapy of extrahepatic diseases on the expressional level. Evasion of liver accumulation and organ-specific targeting and also efficient cytosolic delivery after endosomal internalization are currently insufficiently solved issues. Lipid and polymer-based nanoparticles can be engineered for efficient cellular uptake and enhancement of endosomal escape, but are characterized by preferential liver accumulation based on biodistribution largely determined by particle size and biophysical properties. Oligonucleotide bioconjugates with receptor-binding ligands have been evolved for highly efficient targeting, but frequently result in a large extent of endosomal entrapment and consequently a lack of sufficient cytosolic concentrations. Non-immunoglobulin protein-based receptor recognition affords high cell-type selectivity and is promising for achieving nonhepatic oligonucleotide targeting. The use of such novel protein scaffolds, including designed ankyrin repeat proteins (DARPins), for oligonucleotide delivery is attractive for achieving effective tissue targeting. Issues for further development and optimization to advance approaches for extrahepatic oligonucleotide delivery by nanoparticles or bioconjugates are discussed.
Topics: Animals; Ankyrin Repeat; Endosomes; Gene Transfer Techniques; Humans; Lipids; Liposomes; Muscular Diseases; Nanoparticles; Neoplasms; Oligonucleotides, Antisense; Organ Specificity; Protein Engineering; RNA, Small Interfering; Receptors, Cell Surface; Single-Chain Antibodies
PubMed: 29733239
DOI: 10.1089/nat.2017.0713 -
Frontiers in Plant Science 2020Ankyrin repeat (ANK) proteins are essential in cell growth, development, and response to hormones and environmental stresses. In the present study, 226 genes were...
Ankyrin repeat (ANK) proteins are essential in cell growth, development, and response to hormones and environmental stresses. In the present study, 226 genes were identified and classified into nine subfamilies according to conserved domains in the soybean genome ( L.). Among them, the was highly induced by drought, salt, and abscisic acid. The encodes a protein that belongs to the ANK-RF subfamily containing a RING finger (RF) domain in addition to the ankyrin repeats. Heterologous overexpression of in transgenic improved the germination rate under drought and salt treatments compared to wild-type. Homologous overexpression of improved the survival rate under drought and salt stresses in transgenic soybean hairy roots. In response to drought or salt stress, overexpression in soybean hairy root showed higher proline and lower malondialdehyde contents, and lower HO and O contents compared control plants. Besides, activated transcription of several abiotic stress-related genes, including , , , , and under drought and salt stresses in soybean. These results provide new insights for functional analysis of soybean ANK proteins and will be helpful for further understanding how ANK proteins in plants adapt to abiotic stress.
PubMed: 33193533
DOI: 10.3389/fpls.2020.584167 -
Molecular Reproduction and Development Dec 2021Ankyrin-repeat and SOCS box-containing proteins (ASB) interact with the elongin B-C adapter via their SOCS box domain and with the cullin and ring box proteins to form...
Ankyrin-repeat and SOCS box-containing proteins (ASB) interact with the elongin B-C adapter via their SOCS box domain and with the cullin and ring box proteins to form E3 ubiquitin ligase complexes within the protein ubiquitination pathway. ASB9 in particular is a differentially expressed gene in ovulatory follicles (OFs) induced by the luteinizing hormone (LH) surge or hCG injection in ovarian granulosa cells (GC) while downregulated in growing dominant follicles. Although ASB9 has been involved in biological processes such as protein modification, the signaling network associated with ASB9 in GC is yet to be fully defined. We previously identified and reported ASB9 interactions and binding partners in GC including PAR1, TAOK1, and TNFAIP6/TSG6. Here, we further investigate ASB9 effects on target binding partners regulation and signaling in GC. CRISPR/Cas9-induced inhibition of ASB9 revealed that ASB9 regulates PAR1, TAOK1, TNFAIP6 as well as genes associated with proliferation and cell cycle progression such as PCNA, CCND2, and CCNE2 while CCNA2 was not affected. Inhibition of ASB9 was also associated with increased GC number and decreased caspase3/7 activity, CASP3 expression, and BAX/BCL2 ratio. Furthermore, ASB9 induction in OF in vivo 24 h post-hCG is concomitant with a significant decrease in phosphorylation levels of MAPK3/1 while pMAPK3/1 levels increased following ASB9 inhibition in GC in vitro. Together, these results provide strong evidence for ASB9 as a regulator of GC activity and function by modulating MAPK signaling likely through specific binding partners such as PAR1, therefore controlling GC proliferation and contributing to GC differentiation into luteal cells.
Topics: Ankyrins; Female; Granulosa Cells; Humans; Protein Binding; Suppressor of Cytokine Signaling Proteins; Ubiquitination
PubMed: 34476862
DOI: 10.1002/mrd.23532 -
Mask, the Drosophila ankyrin repeat and KH domain-containing protein, affects microtubule stability.Journal of Cell Science Oct 2021Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and...
Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. In the present study, we demonstrate that the Drosophila ankyrin repeat and KH domain-containing protein Mask negatively affects MT stability in both larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure-function analysis of Mask revealed that its ankyrin repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact on MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the MT-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Taken together, our studies demonstrate that Mask is a novel regulator for MT stability, and such a role of Mask requires normal function of Jupiter.
Topics: Animals; Ankyrin Repeat; DNA-Binding Proteins; Drosophila Proteins; Drosophila melanogaster; Microtubules; Motor Neurons
PubMed: 34553767
DOI: 10.1242/jcs.258512 -
Protein Engineering, Design & Selection... Feb 2021Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior...
Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior work, our group engineered a panel of potent TcdB-neutralizing designed ankyrin repeat proteins (DARPin) as oral therapeutics against C. difficile infection. However, all these DARPins are highly susceptible to digestion by gut-resident proteases, i.e. trypsin and chymotrypsin. Close evaluation of the protein sequence revealed a large abundance of positively charged and aromatic residues in the DARPin scaffold. In this study, we significantly improved the protease stability of one of the DARPins, 1.4E, via protein engineering. Unlike 1.4E, whose anti-TcdB EC50 increased >83-fold after 1-hour incubation with trypsin (1 mg/ml) or chymotrypsin (0.5 mg/ml), the best progenies-T10-2 and T10b-exhibit similar anti-TcdB potency as their parent in PBS regardless of protease treatment. The superior protease stability of T10-2 and T10b is attributed to the removal of nearly all positively charged and aromatic residues except those directly engaged in target binding. Furthermore, T10-2 was found to retain significant toxin-neutralization ability in ex vivo cecum fluid and can be easily detected in mouse fecal samples upon oral administration. Both T10-2 and T10b enjoy a high thermo- and chemo-stability and can be expressed very efficiently in Escherichia coli (>100 mg/l in shaker flasks). We believe that, in additional to their potential as oral therapeutics against C. difficile infection, T10-2 and T10b can also serve as a new generation DARPin scaffold with superior protease stability.
Topics: Animals; Bacterial Proteins; Bacterial Toxins; Clostridioides difficile; Designed Ankyrin Repeat Proteins; Enterotoxins; Mice; Peptide Hydrolases
PubMed: 34882774
DOI: 10.1093/protein/gzab028 -
Annual Review of Pharmacology and... Jan 2020The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology.... (Review)
Review
The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.
Topics: Animals; Ankyrin Repeat; Drug Discovery; Humans; Protein Binding; Protein Engineering; Proteins
PubMed: 31914898
DOI: 10.1146/annurev-pharmtox-010818-021118 -
Biochemical Society Transactions Oct 2015Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular... (Review)
Review
Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular structure that do not fold independently, but rather co-operate in stabilizing structural forms that comprise several repeat-units. For many natural repeat-proteins, it has been shown that weak energetic links between repeats lead to the breakdown of co-operativity and the appearance of folding sub-domains within an apparently regular repeat array. The quasi-1D architecture of repeat-proteins is crucial in detailing how the local energetic balances can modulate the folding dynamics of these proteins, which can be related to the physiological behaviour of these ubiquitous biological systems.
Topics: Animals; Energy Transfer; Evolution, Molecular; Humans; Models, Molecular; Protein Conformation; Protein Folding; Protein Interaction Domains and Motifs; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Repetitive Sequences, Amino Acid; Tandem Repeat Sequences
PubMed: 26517892
DOI: 10.1042/BST20150083 -
Molecular Pharmaceutics Oct 2022Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin,...
Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/ receptor, was site-specifically modified with enzymatic methods and Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and Zr-radiolabeling produced a novel ZrDFO-G3-DARPin radiotracer that can detect HER2/-positive tumors. The triglycine probe, DFO-Gly (), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of ZrDFO-G3-DARPin were measured by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g ( = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, = 5, -value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of ZrDFO-G3-DARPin in HER2/ positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.
Topics: Animals; Cell Line, Tumor; Deferoxamine; Designed Ankyrin Repeat Proteins; Female; Humans; Mice; Mice, Nude; Positron-Emission Tomography; Receptor, ErbB-2; Tissue Distribution; Zirconium
PubMed: 35434995
DOI: 10.1021/acs.molpharmaceut.2c00136