A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis.

BACKGROUND: Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14-3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function. RESULTS: Here we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner. CONCLUSIONS: We found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.

Genome-wide identification of Ankyrin (ANK) repeat gene families in three Dendrobium species and the expression of ANK genes in D. officinale under gibberellin and abscisic acid treatments.

BACKGROUND: Dendrobium Sw. represents one of the most expansive genera within the Orchidaceae family, renowned for its species' high medicinal and ornamental value. In higher plants, the ankyrin (ANK) repeat protein family is characterized by a unique ANK repeat domain, integral to a plethora of biological functions and biochemical activities. The ANK gene family plays a pivotal role in various plant physiological processes, including stress responses, hormone signaling, and growth. Hence, investigating the ANK gene family and identifying disease-resistance genes in Dendrobium is of paramount importance. RESULTS: This research identified 78 ANK genes in Dendrobium officinale Kimura et Migo, 77 in Dendrobium nobile Lindl., and 58 in Dendrobium chrysotoxum Lindl. Subsequently, we conducted comprehensive bioinformatics analyses on these ANK gene families, encompassing gene classification, chromosomal localization, phylogenetic relationships, gene structure and motif characterization, cis-acting regulatory element identification, collinearity assessment, protein-protein interaction network construction, and gene expression profiling. Concurrently, three DoANK genes (DoANK14, DoANK19, and DoANK47) in D. officinale were discerned to indirectly activate the NPR1 transcription factor in the ETI system via SA, thereby modulating the expression of the antibacterial PR gene. Hormonal treatments with GA3 and ABA revealed that 17 and 8 genes were significantly up-regulated, while 4 and 8 genes were significantly down-regulated, respectively. DoANK32 was found to localize to the ArfGAP gene in the endocytosis pathway, impacting vesicle transport and the polar movement of auxin. CONCLUSION: Our findings provide a robust framework for the taxonomic classification, evolutionary analysis, and functional prediction of Dendrobium ANK genes. The three highlighted ANK genes (DoANK14, DoANK19, and DoANK47) from D. officinale may prove valuable in disease resistance and stress response research. DoANK32 is implicated in the morphogenesis and development of D. officinale through its role in vesicular transport and auxin polarity, with subcellular localization studies confirming its presence in the nucleus and cell membrane. ANK genes displaying significant expression changes in response to hormonal treatments could play a crucial role in the hormonal response of D. officinale, potentially inhibiting its growth and development through the modulation of plant hormones such as GA3 and ABA.

The RXLR effector PpE18 of Phytophthora parasitica is a virulence factor and suppresses peroxisome membrane-associated ascorbate peroxidase NbAPX3-1-mediated plant immunity.

Phytophthora parasitica causes diseases on a broad range of host plants. It secretes numerous effectors to suppress plant immunity. However, only a few virulence effectors in P. parasitica have been characterized. Here, we highlight that PpE18, a conserved RXLR effector in P. parasitica, was a virulence factor and suppresses Nicotiana benthamiana immunity. Utilizing luciferase complementation, co-immunoprecipitation, and GST pull-down assays, we determined that PpE18 targeted NbAPX3-1, a peroxisome membrane-associated ascorbate peroxidase with reactive oxygen species (ROS)-scavenging activity and positively regulates plant immunity in N. benthamiana. We show that the ROS-scavenging activity of NbAPX3-1 was critical for its immune function and was hindered by the binding of PpE18. The interaction between PpE18 and NbAPX3-1 resulted in an elevation of ROS levels in the peroxisome. Moreover, we discovered that the ankyrin repeat-containing protein NbANKr2 acted as a positive immune regulator, interacting with both NbAPX3-1 and PpE18. NbANKr2 was required for NbAPX3-1-mediated disease resistance. PpE18 competitively interfered with the interaction between NbAPX3-1 and NbANKr2, thereby weakening plant resistance. Our results reveal an effective counter-defense mechanism by which P. parasitica employed effector PpE18 to suppress host cellular defense, by suppressing biochemical activity and disturbing immune function of NbAPX3-1 during infection.

Redesigning amino/carboxyl ends of DARPin G3 for high thermostability and production in tobacco transplastomic plants.

KEY MESSAGE: Redesigning the N- and C-capping repeats of the native DARPin G3 significantly improved its stability, and may facilitate its purification from the total soluble proteins of high-temperature dried leaf materials of transplastomic plants. Designed ankyrin repeat proteins (DARPins) constitute a promising class of binding molecules that can overcome the limitations of monoclonal antibodies and enable the development of novel therapeutic approaches. Despite their inherent stability, detailed studies have revealed that the original capping repeats derived from natural ankyrin repeat proteins impair the stability of the initial DARPin design. Consequently, the development of thermodynamically stabilized antibody mimetics may facilitate the development of innovative drugs in the future. In this study, we replaced the original N- and C-capping repeats with improved caps to enhance the thermostability of native DARPin G3. Computational analyses suggested that the redesigned thermostable DARPin G3 structure possessed optimal quality and stability. Molecular dynamics simulations verified the stability of the redesigned thermostable DARPin G3 at high temperatures. The redesigned thermostable DARPin G3 was expressed at high levels in tobacco transplastomic plants and subsequently purified from high-temperature dried leaf materials. Thermal denaturation results revealed that the redesigned thermostable DARPin G3 had a higher Tm value than the native DARPin G3, with a Tm of 35.51 °C greater than that of native DARPin G3. The results of the in vitro bioassays confirmed that the purified thermostable DARPin G3 from high-temperature dried leaf materials maintained its binding activity without any loss of affinity and specifically bound to the HER2 receptor on the cell surface. These findings demonstrate the successful improvement in the thermostability of DARPin G3 without compromising its biological activity.

Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

GmANKTM21 Positively Regulates Drought Tolerance and Enhanced Stomatal Response through the MAPK Signaling Pathway in Soybean.

Drought stress is one of the significant abiotic stresses that limit soybean (Glycine max [L.] Merr.) growth and production. Ankyrin repeat (ANK) proteins, being highly conserved, occupy a pivotal role in diverse biological processes. ANK genes were classified into nine subfamilies according to conserved domains in the soybean genome. However, the function of ANK-TM subfamily proteins (Ankyrin repeat proteins with a transmembrane domain) in the abiotic-stress response to soybean remains poorly understood. In this study, we first demonstrated the subcellular localization of GmANKTM21 in the cell membrane and nucleus. Drought stress-induced mRNA levels of GmANKTM21, which encodes proteins belonging to the ANK-TM subfamily, Transgenic 35S:GmANKTM21 soybean improved drought tolerance at the germination and seedling stages, with higher stomatal closure in soybean, lower water loss, lower malondialdehyde (MDA) content, and less reactive oxygen species (ROS) production compared with the wild-type soybean (Dongnong50). RNA-sequencing (RNA-seq) and RT-qPCR analysis of differentially expressed transcripts in overexpression of GmANKTM21 further identified potential downstream genes, including GmSPK2, GmSPK4, and GmCYP707A1, which showed higher expression in transgenic soybean, than those in wild-type soybean and KEGG enrichment analysis showed that MAPK signaling pathways were mostly enriched in GmANKTM21 overexpressing soybean plants under drought stress conditions. Therefore, we demonstrate that GmANKTM21 plays an important role in tolerance to drought stress in soybeans.

X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Šresolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo' proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

Unique ankyrin repeat proteins in the genome of poxviruses-Boon or Wane, a critical review.

Ankyrin repeat is a 33-amino acid motif commonly observed in eukaryotes and, to a lesser extent, in prokaryotes and archaea and rarely in viruses. This motif plays a crucial role in regulating various cellular processes like the cell cycle, transcription, cell signaling, and inflammatory responses through interactions between proteins. Poxviruses exhibit a distinctive feature of containing multiple ankyrin repeat proteins within their genomes. All the genera of poxviruses possess these proteins except molluscipox virus, crocodylidpox virus, and red squirrel poxvirus. An intriguing characteristic has generated notable interest in studying the functions of these proteins within poxvirus biology. Within poxviruses, ankyrin repeat proteins exhibit a distinct configuration, featuring ankyrin repeats in the N-terminal region and a cellular F-box homolog in the C-terminal region, which enables interactions with the cellular Skp, Cullin, F-box containing ubiquitin ligase complex. Through the examination of experimental evidences and discussions from current literature, this review elucidates the organization and role of ankyrin repeat proteins in poxviruses. Various research studies have highlighted the significant importance of these proteins in poxviral pathogenesis and, acting as factors that enhance virulence. Consequently, they represent viable targets for developing genetically altered viruses with decreased virulence, thus displaying potential as candidates for vaccines and antiviral therapeutic development contributing to safer and more effective strategies against poxviral infections.

Deletion of Asb15b gene can lead to a significant decrease in zebrafish intermuscular bone.

Intermuscular bones, which are present in numerous economically significant fish species, have a negative impact on the development of aquaculture. The Asb15b gene, primarily expressed in skeletal muscle, plays a crucial role in regulating protein turnover and the development of muscle fibers. It stimulates protein synthesis and controls the differentiation of muscle fibers. In this study, we employed CRISPR/Cas9 technology to generate homozygous zebrafish strains with 7 bp and 49 bp deletions in the Asb15b gene. Subsequent analyses using skeleton staining demonstrated a substantial reduction in the number of intermuscular bones in adult Asb15b-/- -7 bp and Asb15b-/- -49 bp mutants compared to the wild-type zebrafish, with decreases of 30 % (P < 0.001) and 40 % (P < 0.0001), respectively. Histological experiments further revealed that the diameter and number of muscle fibers in adult Asb15b-/- mutants did not exhibit significant changes when compared to wild-type zebrafish. Moreover, qRT-PCR experiments demonstrated significant differences in the expression of bmp6 and runx2b genes, which are key regulators of intermuscular bone development, during different stages of intermuscular bone development in Asb15b-/- mutants. This study strongly suggests that the Asb15b gene plays a crucial role in regulating intermuscular bone development in fish and lays the groundwork for further exploration of the role of the Asb15b gene in zebrafish intermuscular bone development.

Honokiol targeting ankyrin repeat domain of TRPV4 ameliorates endothelial permeability in mice inflammatory bowel disease induced by DSS.

ETHNOPHARMACOLOGICAL RELEVANCE: As a classic traditional Chinese medicine, Magnolia officinalis (M. officinalis) is widely used in digestive diseases. It has rich gastrointestinal activity including inflammatory bowel disease (IBD) treatment, but the mechanism is not clear. AIM OF THE STUDY: In recent years, there has been a growing interest in investigating the regulatory effects of herbal compounds on transient receptor potential (TRP) channel proteins. Transient receptor potential vanilloid 4 (TRPV4), a subtype involved in endothelial permeability regulation, was discussed as the target of M. officinalis in the treatment of IBD in the study. Based on the targeting effect of TRPV4, this study investigated the active ingredients and mechanism of M. officinalis extract in treating IBD. MATERIALS AND METHODS: To reveal the connection between the active ingredients in M. officinalis and TRPV4, a bioactivity-guided high performance liquid chromatography system coupled with mass spectrometry identification was utilized to screen for TRPV4 antagonists. TRPV4 siRNA knockdown experiment was employed to validate the significance of TRPV4 as a crucial target in regulating endothelial permeability by honokiol (HON). The interaction of the active ingredient representing HON with TRPV4 was confirmed by molecular docking, fluorescence-based thermal shift and live cell calcium imaging experiments. The potential binding sites and inhibitory mechanisms of HON in TRPV4 were analyzed by molecular dynamics simulation and microscale thermophoresis. The therapeutic effect of HON based on TRPV4 was discussed in DSS-IBD mice. RESULTS: Our finding elucidated that the inhibitory activity of M. officinalis against TRPV4 is primarily attributed to HON analogues. The knockdown of TRPV4 expression significantly impaired the calcium regulation and permeability protection in endothelial cells. The mechanism study revealed that HON specifically targets the Q239 residue located in the ankyrin repeat domain of TRPV4, and competitively inhibits channel opening with adenosine triphosphate (ATP) binding. The immunofluorescence assay demonstrated that the administration of HON enhances the expression and location of VE-Cadherin to protect the endothelial barrier and attenuates immune cell infiltration. CONCLUSIONS: The finding suggested that HON alleviates IBD by improving endothelial permeability through TRPV4. The discovery provides valuable insights into the potential therapeutic strategy of active natural products for alleviating IBD.

Molecular characterization and functional analysis of Eimeria tenella ankyrin repeat-containing protein.

Chicken coccidiosis causes disastrous losses to the poultry industry all over the world. Eimeria tenella is the most prevalent of these disease-causing species. Our former RNA-seq indicated that E. tenella ankyrin repeat-containing protein (EtANK) was expressed differently between drug-sensitive (DS) and drug-resistant strains. In this study, we cloned EtANK and analyzed its translational and transcriptional levels using quantitative real-time PCR (qPCR) and western blotting. The data showed that EtANK was significantly upregulated in diclazuril-resistant (DZR) strain and maduramicin-resistant (MRR) strain compared with the drug-sensitive (DS) strain. In addition, the transcription levels in the DZR strains isolated from the field were higher than in the DS strain. The translation levels of EtANK were higher in unsporulated oocysts (UO) than in sporozoites (SZ), sporulated oocysts (SO), or second-generation merozoites (SM), and the protein levels in SM were significantly higher than in UO, SO, and SZ. The results of the indirect immunofluorescence localization showed that the protein was distributed mainly at the anterior region of SZ and on the surface and in the cytoplasm of SM. The fluorescence intensity increased further with its development in vitro. An anti-rEtANK polyclonal antibody inhibited the invasive ability of E. tenella in DF-1 cells. These results showed that EtANK may be related to host cell invasion, required for the parasite's growth in the host, and may be involved in the development of E. tenella resistance to some drugs.

ANKZF1 knockdown inhibits glioblastoma progression by promoting intramitochondrial protein aggregation through mitoRQC.

Protein homeostasis is fundamental to the development of tumors. Ribosome-associated quality-control (RQC) is able to add alanine and threonine to the stagnant polypeptide chain C-terminal (CAT-tail) when protein translation is hindered, while Ankyrin repeat and zinc-finger domain-containing-protein 1 (ANKZF1) can counteract the formation of the CAT-tail, preventing the aggregation of polypeptide chains. In particular, ANKZF1 plays an important role in maintaining mitochondrial protein homeostasis by mitochondrial RQC (mitoRQC) after translation stagnation of precursor proteins targeting mitochondria. However, the role of ANKZF1 in glioblastoma is unclear. Therefore, the current study was aimed to investigate the effects of ANKZF1 in glioblastoma cells and a nude mouse glioblastoma xenograft model. Here, we reported that knockdown of ANKZF1 in glioblastoma cells resulted in the accumulation of CAT-tail in mitochondria, leading to the activated mitochondrial unfolded protein response (UPRmt) and inhibits glioblastoma malignant progression. Excessive CAT-tail sequestered mitochondrial chaperones HSP60, mtHSP70 and proteases LONP1 as well as mitochondrial respiratory chain subunits ND1, Cytb, mtCO2 and ATP6, leading to mitochondrial oxidative phosphorylation dysfunction, membrane potential impairment, and mitochondrial apoptotic pathway activation. Our study highlights ANKZF1 as a valuable target for glioblastoma intervention and provides an innovative insight for the treatment of glioblastoma through the regulating of mitochondrial protein homeostasis.