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PloS One 2023Interleukin (IL)-27, a member of the IL-12 family of cytokines, induces human immunodeficiency virus (HIV)-resistant monocyte-derived macrophages and T cells. This...
Interleukin (IL)-27, a member of the IL-12 family of cytokines, induces human immunodeficiency virus (HIV)-resistant monocyte-derived macrophages and T cells. This resistance is mediated via the downregulation of spectrin beta, non-erythrocytic 1 (SPTBN1), induction of autophagy, or suppression of the acetylation of Y-box binding protein-1 (YB-1); however, the role of IL-27 administration during the induction of immature monocyte-derived dendritic cells (iDC) is poorly investigated. In the current study, we investigated the function of IL-27-induced iDC (27DC) on HIV infection. 27DC inhibited HIV infection by 95 ± 3% without significant changes in the expression of CD4, CCR5, and SPTBN1 expression, autophagy induction and acetylation of YB-1 compared to iDC. An HIV proviral DNA copy number assay displayed that 27DC suppressed reverse transcriptase (RT) reaction without influencing the virus entry. A DNA microarray analysis was performed to identify the differentially expressed genes between 27DC and iDC. Compared to iDC, 51 genes were differentially expressed in 27DC, with more than 3-fold changes in four independent donors. Cross-reference analysis with the reported 2,214 HIV regulatory host genes identified nine genes as potential interests: Ankyrin repeat domain 22, Guanylate binding protein (GBP)-1, -2, -4, -5, Stabilin 1, Serpin family G member 1 (SERPING1), Interferon alpha inducible protein 6, and Interferon-induced protein with tetratricopeptide repeats 3. A knock-down study using si-RNA failed to determine a key factor associated with the anti-HIV activity due to the induction of robust amounts of off-target effects. Overexpression of each protein in cells had no impact on HIV infection. Thus, we could not define the mechanism of the anti-HIV effect in 27DC. However, our findings indicated that IL-27 differentiates monocytes into HIV-resistant DC, and the inhibitory mechanism differs from IL-27-induced HIV-resistant macrophages and T cells.
Topics: Humans; Interleukin-27; HIV Infections; Virus Internalization; HIV-1; Interleukins; Monocytes; Autophagy; DNA; Dendritic Cells; Virus Replication; Spectrin
PubMed: 37910521
DOI: 10.1371/journal.pone.0287829 -
Genes Oct 2023The KN Motif and AnKyrin Repeat Domain 1 () is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and...
The KN Motif and AnKyrin Repeat Domain 1 () is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for in lymphoid cell development, and total protein analysis suggests the involvement of in BM cells' cytoskeleton formation and mobility.
Topics: Animals; Humans; Mice; Adaptor Proteins, Signal Transducing; Ankyrin Repeat; Cytoskeletal Proteins; Disease Models, Animal; Neoplasms
PubMed: 37895296
DOI: 10.3390/genes14101947 -
Scientific Reports Oct 2023Glucagon-like peptide-1 (GLP-1) is an intestinally derived incretin that plays a vital role in engineering the biological circuit involved in treating type 2 diabetes....
Glucagon-like peptide-1 (GLP-1) is an intestinally derived incretin that plays a vital role in engineering the biological circuit involved in treating type 2 diabetes. Exceedingly short half-life (1-2 min) of GLP-1 limits its therapeutic applicability, and the implication of its new variants is under question. Since albumin-binding DARPin as a mimetic molecule has been reported to increase the serum half-life of therapeutic compounds, the interaction of new variants of GLP-1 in fusion with DARPin needs to be examined against the GLP-1 receptor. This study was aimed to design stable and functional fusion proteins consisting of new protease-resistant GLP-1 mutants (mGLP1) genetically fused to DARPin as a critical step toward developing long-acting GLP-1 receptor agonists. The stability and solubility of the engineered fusion proteins were analyzed, and their secondary and tertiary structures were predicted and satisfactorily validated. Molecular dynamics simulation studies revealed that the predicted structures of engineered fusion proteins remained stable throughout the simulation. The relative binding affinity of the engineered fusion proteins' complex with human serum albumin and the GLP-1 receptor individually was assessed using molecular docking analyses. It revealed a higher affinity compared to the interaction of the individual GLP-1 and HSA-binding DARPin with the GLP-1 receptor and human serum albumin, respectively. The present study suggests that engineered fusion proteins can be used as a potential molecule in the treatment of type 2 diabetes, and this study provides insight into further experimental use of mimetic complexes as alternative molecules to be evaluated as new bio-breaks in the engineering of biological circuits in the treatment of type 2 diabetes.
Topics: Humans; Designed Ankyrin Repeat Proteins; Diabetes Mellitus, Type 2; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Hypoglycemic Agents; Molecular Docking Simulation; Serum Albumin, Human
PubMed: 37853095
DOI: 10.1038/s41598-023-45185-1 -
PLoS Computational Biology Oct 2023TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided...
TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided numerous insights into the structural rearrangements accompanying their opening and closing; however, the molecular mechanisms by which TRPV channels establish precise and robust temperature sensing remain elusive. In this work we employ molecular simulations, multi-ensemble contact analysis, graph theory, and machine learning techniques to reveal the temperature-sensitive residue-residue interactions driving allostery in TRPV3. We find that groups of residues exhibiting similar temperature-dependent contact frequency profiles cluster at specific regions of the channel. The dominant mode clusters on the ankyrin repeat domain and displays a linear melting trend while others display non-linear trends. These modes describe the residue-level temperature response patterns that underlie the channel's functional dynamics. With network analysis, we find that the community structure of the channel changes with temperature. And that a network of high centrality contacts connects distant regions of the protomer to the gate, serving as a means for the temperature-sensitive contact modes to allosterically regulate channel gating. Using a random forest model, we show that the contact states of specific temperature-sensitive modes are indeed predictive of the channel gate's state. Supporting the physical validity of these modes and networks are several residues identified with our analyses that are reported in literature to be functionally critical. Our results offer high resolution insight into thermo-TRP channel function and demonstrate the utility of temperature-sensitive contact analysis.
Topics: Temperature; Protein Subunits; Ankyrin Repeat
PubMed: 37831724
DOI: 10.1371/journal.pcbi.1011545 -
Cell Death & Disease Oct 2023The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of...
The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p63/p73 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p63/p73 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.
Topics: Animals; Humans; Mice; Carcinoma, Squamous Cell; Designed Ankyrin Repeat Proteins; Transcription Factors; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins
PubMed: 37828008
DOI: 10.1038/s41419-023-06213-0 -
Emerging Microbes & Infections Dec 2023Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear...
Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.
Topics: Animals; Humans; Nucleoproteins; Viral Proteins; Influenza A virus; Viral Core Proteins; Protein Binding; Virus Replication; Repressor Proteins
PubMed: 37823597
DOI: 10.1080/22221751.2023.2270073 -
Journal of Neuroscience Methods Nov 2023Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain...
BACKGROUND
Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies.
METHODS
Two alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells.
RESULTS
Compared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons.
CONCLUSION
The substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.
Topics: Rats; Animals; Genetic Vectors; Dependovirus; Gene Transfer Techniques; Cell Line; Genetic Therapy; Transduction, Genetic
PubMed: 37783350
DOI: 10.1016/j.jneumeth.2023.109981 -
NPJ Vaccines Sep 2023Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing...
Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing.
PubMed: 37777519
DOI: 10.1038/s41541-023-00746-3 -
International Journal of Molecular... Sep 2023Breast cancer is a major global health burden with high morbidity and mortality rates. Previous studies have reported that increased expression of ASAP1 is associated...
Breast cancer is a major global health burden with high morbidity and mortality rates. Previous studies have reported that increased expression of ASAP1 is associated with poor prognosis in various types of cancer. This study was conducted on 452 breast cancer patients who underwent surgery at Hanyang University Hospital, Seoul, South Korea. Data on clinicopathological characteristics including molecular pathologic markers were collected. Immunohistochemical staining of ASAP1 expression level were used to classify patients into high and low groups. In total, 452 cases low ASAP1 expression group was associated with significantly worse recurrence-free survival ( = 0.029). In ER-positive cases ( = 280), the low ASAP1 expression group was associated with significantly worse overall survival ( = 0.039) and recurrence-free survival ( = 0.029). In multivariate cox analysis, low ASAP1 expression was an independent significant predictor of poor recurrence-free survival in the overall patient group (hazard ratio = 2.566, = 0.002) and ER-positive cases (hazard ratio = 4.046, = 0.002). In the analysis of the TCGA dataset, the low-expression group of ASAP1 protein demonstrated a significantly poorer progression-free survival ( = 0.005). This study reports that low ASAP1 expression was associated with worse recurrence-free survival in invasive breast cancer.
Topics: Humans; Female; Breast Neoplasms; Prognosis; Hospitals, University; Multivariate Analysis; Progression-Free Survival; Adaptor Proteins, Signal Transducing
PubMed: 37762658
DOI: 10.3390/ijms241814355 -
Molecular Brain Sep 2023Novelty-induced memory consolidation is a well-established phenomenon that depends on the activation of a locus coeruleus-hippocampal circuit. It is associated with the...
Novelty-induced memory consolidation is a well-established phenomenon that depends on the activation of a locus coeruleus-hippocampal circuit. It is associated with the expression of activity-dependent genes that may mediate initial or cellular memory consolidation. Several genes have been identified to date, however, to fully understand the mechanisms of memory consolidation, additional candidates must be identified. In this cross-species study, we used a contextual novelty-exploration paradigm to identify changes in gene expression in the dorsal hippocampus of both mice and rats. We found that changes in gene expression following contextual novelty varied between the two species, with 9 genes being upregulated in mice and 3 genes in rats. Comparison across species revealed that ArfGAP with a GTPase domain, an ankyrin repeat and PH domain 3 (Agap3) was the only gene being upregulated in both, suggesting a potentially conserved role for Agap3. AGAP3 is known to regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor trafficking in the synapse, which suggests that increased transcription of Agap3 may be involved in maintaining functional plasticity. While we identified several genes affected by contextual novelty exploration, we were unable to fully reverse these changes using SCH 23390, a dopamine D/D receptor antagonist. Further research on the role of AGAP3 in novelty-induced memory consolidation could lead to better understanding of this process and guide future research.
Topics: Animals; Mice; Rats; Dopamine; Glutamic Acid; Hippocampus; Locus Coeruleus; Memory Consolidation; Receptors, AMPA; GTPase-Activating Proteins
PubMed: 37749596
DOI: 10.1186/s13041-023-01056-4