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Molecular and Cellular Biology 2024Cellular senescence is a dynamic biological process triggered by sublethal cell damage and driven by specific changes in gene expression programs. We recently identified...
Cellular senescence is a dynamic biological process triggered by sublethal cell damage and driven by specific changes in gene expression programs. We recently identified ANKRD1 (ankyrin repeat domain 1) as a protein strongly elevated after triggering senescence in fibroblasts. Here, we set out to investigate the mechanisms driving the elevated production of ANKRD1 in the early stages of senescence. Our results indicated that the rise in ANKRD1 levels after triggering senescence using etoposide (Eto) was the result of moderate increases in transcription and translation, and robust mRNA stabilization. Antisense oligomer (ASO) pulldown followed by mass spectrometry revealed a specific interaction of the RNA-binding protein RBMS1 with mRNA that was confirmed by ribonucleoprotein immunoprecipitation analysis. RBMS1 abundance decreased in the nucleus and increased in the cytoplasm during Eto-induced senescence; in agreement with the hypothesis that RBMS1 may participate in post-transcriptional stabilization of mRNA, silencing RBMS1 reduced, while overexpressing RBMS1 enhanced mRNA half-life after Eto treatment. A segment proximal to the coding region was identified as binding RBMS1 and conferring RBMS1-dependent increased expression of a heterologous reporter. We propose that RBMS1 increases expression of ANKRD1 during the early stages of senescence by stabilizing mRNA.
Topics: Humans; Cellular Senescence; RNA Stability; RNA-Binding Proteins; Nuclear Proteins; Repressor Proteins; RNA, Messenger; Etoposide; Fibroblasts; Cell Nucleus; Cell Line; Muscle Proteins
PubMed: 38769646
DOI: 10.1080/10985549.2024.2350540 -
Acta Crystallographica. Section F,... May 2024Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by...
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 Å resolution. 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.
Topics: Crystallography, X-Ray; Models, Molecular; Ankyrin Repeat; Cryoelectron Microscopy; Ligands; Protein Conformation; Protein Binding; Gene Expression
PubMed: 38767964
DOI: 10.1107/S2053230X2400414X -
Gene Sep 2024Intermuscular bones, which are present in numerous economically significant fish species, have a negative impact on the development of aquaculture. The Asb15b gene,...
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.
Topics: Animals; Bone and Bones; Bone Development; CRISPR-Cas Systems; Gene Deletion; Gene Expression Regulation, Developmental; Muscle, Skeletal; Zebrafish; Zebrafish Proteins; Ankyrin Repeat
PubMed: 38754570
DOI: 10.1016/j.gene.2024.148561 -
European Journal of Protistology Jun 2024Chicken coccidiosis causes disastrous losses to the poultry industry all over the world. Eimeria tenella is the most prevalent of these disease-causing species. Our...
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.
Topics: Eimeria tenella; Protozoan Proteins; Ankyrin Repeat; Animals; Triazines; Chickens; Coccidiostats; Nitriles; Drug Resistance; Coccidiosis; Poultry Diseases; Benzamides; Lactones
PubMed: 38749182
DOI: 10.1016/j.ejop.2024.126089 -
International Journal of Molecular... Apr 2024, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Kuwayama, the vector of Liberibacter asiaticus (Las) that causes...
, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Kuwayama, the vector of Liberibacter asiaticus (Las) that causes citrus greening disease, is naturally infected with (Di). However, the interaction between and remains poorly understood. In this study, we performed a pan-genome analysis using 65 Di genomes to gain a comprehensive understanding of Di. Based on average nucleotide identity (ANI) analysis, we classified the Di strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed Di genomes. The variation analysis between Asia and North America Di showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of Di contributes to our understanding of the geographical population of Di, the origin of hosts of , and the interaction between Di and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).
Topics: Wolbachia; Symbiosis; Genome, Bacterial; Animals; Phylogeny; Asia; North America; Hemiptera; Diptera; Polymorphism, Single Nucleotide
PubMed: 38732070
DOI: 10.3390/ijms25094851 -
Journal of Cancer 2024This study investigated the significance of HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) in esophageal cancer (ESCA) and its...
This study investigated the significance of HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) in esophageal cancer (ESCA) and its underlying mechanism in ESCA regulation through the induction of RAC1 ubiquitination and degradation. Characterization studies of HACE1 in ESCA clinical tissues and cell lines were performed. Next, the effects of HACE1 on the biological behavior of ESCA cells were examined by silencing and overexpressing HACE1. Protein-protein interactions (PPIs) involving HACE1 were analyzed using data from the String website. The function of HACE1 in RAC1 protein ubiquitination was validated using the proteasome inhibitor MG132. The effects of HACE1 on ESCA cells through RAC1 were elucidated by applying the RAC1 inhibitor EHop-016 in a tumor-bearing nude mouse model. To establish the relationship between HACE1 and TRIP12, rescue experiments were conducted, mainly to evaluate the effect of TRIP12 silencing on HACE1-mediated RAC1 regulation in vitro and in vivo. The PPI between HACE1 and TRIP12 and their subcellular localization were further characterized through co-immunoprecipitation and immunofluorescence staining assays, respectively. HACE1 protein expression was notably diminished in ESCA cells but upregulated in normal tissues. HACE1 overexpression inhibited the malignant biological behavior of ESCA cells, leading to restrained tumor growth in mice. This effect was coupled with the promotion of RAC1 protein ubiquitination and subsequent degradation. Conversely, silencing HACE1 exhibited contrasting results. PPI existed between HACE1 and TRIP12, compounded by their similar subcellular localization. Intriguingly, TRIP12 inhibition blocked HACE1-driven RAC1 ubiquitination and mitigated the inhibitory effects of HACE1 on ESCA cells, alleviating tumor growth in the tumor-bearing nude mouse model. HACE1 expression was downregulated in ESCA cells, suggesting that it curbs ESCA progression by inducing RAC1 protein degradation through TRIP12-mediated ubiquitination.
PubMed: 38706891
DOI: 10.7150/jca.93833 -
Disease Models & Mechanisms Apr 2024Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is... (Review)
Review
Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is increasingly implicated in human disease. ANKLE2 regulates nuclear envelope disassembly at the onset of mitosis and its reassembly after chromosome segregation. ANKLE2 dysfunction is associated with abnormal nuclear morphology and cell division. It regulates the nuclear envelope by mediating protein-protein interactions with barrier to autointegration factor (BANF1; also known as BAF) and with the kinase and phosphatase that modulate the phosphorylation state of BAF. In brain development, ANKLE2 is crucial for proper asymmetric division of neural progenitor cells. In humans, pathogenic loss-of-function mutations in ANKLE2 are associated with primary congenital microcephaly, a condition in which the brain is not properly developed at birth. ANKLE2 is also linked to other disease pathologies, including congenital Zika syndrome, cancer and tauopathy. Here, we review the molecular roles of ANKLE2 and the recent literature on human diseases caused by its dysfunction.
Topics: Humans; Nuclear Proteins; Animals; Disease; DNA-Binding Proteins; Mutation
PubMed: 38691001
DOI: 10.1242/dmm.050554 -
Journal of Muscle Research and Cell... Apr 2024The muscular dystrophy with myositis (mdm) mouse model results in a severe muscular dystrophy due to an 83-amino-acid deletion in the N2A region of titin, an expanded...
The muscular dystrophy with myositis (mdm) mouse model results in a severe muscular dystrophy due to an 83-amino-acid deletion in the N2A region of titin, an expanded sarcomeric protein that functions as a molecular spring which senses and modulates the response to mechanical forces in cardiac and skeletal muscles. ANKRD1 is one of the muscle ankyrin repeat domain proteins (MARPs) a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. The aberrant over-activation of Nuclear factor Kappa B (NF-κB) and the Ankyrin-repeat domain containing protein 1 (ANKRD1) occurs in several models of progressive muscle disease including Duchenne muscular dystrophy. We hypothesized that mechanical regulation of ANKRD1 is mediated by NF-κB activation in skeletal muscles and that this mechanism is perturbed by small deletion of the stretch-sensing titin N2A region in the mdm mouse. We applied static mechanical stretch of the mdm mouse diaphragm and cyclic mechanical stretch of CC myotubes to examine the interaction between NF-κΒ and ANKRD1 expression utilizing Western blot and qRTPCR. As seen in skeletal muscles of other severe muscular dystrophies, an aberrant increased basal expression of NF-κB and ANKRD1 were observed in the diaphragm muscles of the mdm mice. Our data show that in the mdm diaphragm, basal levels of NF-κB are increased, and pharmacological inhibition of NF-κB does not alter basal levels of ANKRD1. Alternatively, NF-κB inhibition did alter stretch-induced ANKRD1 upregulation. These data show that NF-κB activity is at least partially responsible for the stretch-induced expression of ANKRD1.
PubMed: 38683293
DOI: 10.1007/s10974-024-09671-x -
Cancer Immunology Research Jul 2024The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC)...
The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).
Topics: Humans; Leukemia, Myeloid, Acute; Animals; Mice; Neoplastic Stem Cells; T-Lymphocytes; Interleukin-3 Receptor alpha Subunit; Xenograft Model Antitumor Assays; Sialic Acid Binding Ig-like Lectin 3; CD3 Complex; Cell Line, Tumor; Cytotoxicity, Immunologic
PubMed: 38683145
DOI: 10.1158/2326-6066.CIR-23-0692 -
Plants (Basel, Switzerland) Apr 2024Plant acyl-CoA-binding proteins (ACBPs) function in plant development and stress responses, with some ACBPs interacting with protein partners. This study tested the...
Plant acyl-CoA-binding proteins (ACBPs) function in plant development and stress responses, with some ACBPs interacting with protein partners. This study tested the interaction between two Class II GmACBPs ( ACBPs) and seven kinases, using yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC). The results revealed that both GmACBP3.1 and GmACBP4.1 interact with two soybean kinases, a mitogen-activated protein kinase MPK2, and a serine/threonine-protein kinase SAPK2, highlighting the significance of the ankyrin-repeat (ANK) domain in facilitating protein-protein interactions. Moreover, an kinase assay and subsequent Phos-tag SDS-PAGE determined that GmMPK2 and GmSAPK2 possess the ability to phosphorylate Class II GmACBPs. Additionally, the kinase-specific phosphosites for Class II GmACBPs were predicted using databases. The HDOCK server was also utilized to predict the binding models of Class II GmACBPs with these two kinases, and the results indicated that the affected residues were located in the ANK region of Class II GmACBPs in both docking models, aligning with the findings of the Y2H and BiFC experiments. This is the first report describing the interaction between Class II GmACBPs and kinases, suggesting that Class II GmACBPs have potential as phospho-proteins that impact signaling pathways.
PubMed: 38674555
DOI: 10.3390/plants13081146