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The Biochemical Journal Feb 2024Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and...
Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.
Topics: Protein Serine-Threonine Kinases; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Leucine-Rich Repeat Proteins; Protein Domains; Mutation; Peptides; Phosphorylation
PubMed: 38305364
DOI: 10.1042/BCJ20230477 -
Journal of Translational Autoimmunity Jun 2024Acute respiratory distress syndrome (ARDS) is independently associated with a poor prognosis in patients with sepsis. Macrophage M1 polarization plays an instrumental...
Acute respiratory distress syndrome (ARDS) is independently associated with a poor prognosis in patients with sepsis. Macrophage M1 polarization plays an instrumental role in this process. Therefore, the exploration of key molecules affecting acute lung injury and macrophage M1 polarization may provide therapeutic targets for the treatment of septic ARDS. Here, we identified that elevated levels of Ankyrin repeat domain-containing protein 22 (ANKRD22) were associated with poor prognosis and more pronounced M1 macrophage polarization in septic patients by analyzing high-throughput data. ANKRD22 expression was also significantly upregulated in the alveolar lavage fluid, peripheral blood, and lung tissue of septic ARDS model mice. Knockdown of ANKRD22 significantly attenuated acute lung injury in mice with sepsis-induced ARDS and reduced the M1 polarization of lung macrophages. Furthermore, deletion of ANKRD22 in macrophages inhibited M1 macrophage polarization and reduced levels of phosphorylated IRF3 and intracellular interferon regulatory factor 3 (IRF3) expression, while re-expression of ANKRD22 reversed these changes. Further experiments revealed that ANKRD22 promotes IRF3 activation by binding to mitochondrial antiviral-signaling protein (MAVS). In conclusion, these findings suggest that ANKRD22 promotes the M1 polarization of lung macrophages and exacerbates sepsis-induced ARDS.
PubMed: 38225946
DOI: 10.1016/j.jtauto.2023.100228 -
Cancer Letters Mar 2024Cervical cancer (CC) patients with lymph node metastasis (LNM) have a poor prognosis. However, the molecular mechanism of LNM in CC is unclear, and there is no effective...
Cervical cancer (CC) patients with lymph node metastasis (LNM) have a poor prognosis. However, the molecular mechanism of LNM in CC is unclear, and there is no effective clinical treatment. Here, we found that 7-dehydrocholesterol reductase (DHCR7), an enzyme that catalyzes the last step of cholesterol synthesis, was upregulated in CC and closely related to LNM. Gain-of-function and loss-of-function experiments proved that DHCR7 promoted the invasion ability of CC cells and lymphangiogenesis in vitro and induced LNM in vivo. The LNM-promoting effect of DHCR7 was partly mediated by upregulating KN motif and ankyrin repeat domains 4 (KANK4) expression and subsequently activating the PI3K/AKT signaling pathway. Alternatively, DHCR7 promoted the secretion of vascular endothelial growth factor-C (VEGF-C), and thereby lymphangiogenesis. Interestingly, cholesterol reprogramming was needed for the DHCR7-mediated promotion of activation of the KANK4/PI3K/AKT axis, VEGF-C secretion, and subsequent LNM. Importantly, treatment with the DHCR7 inhibitors AY9944 and tamoxifen (TAM) significantly inhibited LNM of CC, suggesting the clinical application potential of DHCR7 inhibitors in CC. Collectively, our results uncover a novel molecular mechanism of LNM in CC and identify DHCR7 as a new potential therapeutic target.
Topics: Female; Humans; Cholesterol; Lymphangiogenesis; Lymphatic Metastasis; Oxidoreductases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor C
PubMed: 38211648
DOI: 10.1016/j.canlet.2024.216609 -
PNAS Nexus Jan 2024Mitochondrial features and activities vary in a cell type- and developmental stage-dependent manner to critically impact cell function and lineage development....
Mitochondrial features and activities vary in a cell type- and developmental stage-dependent manner to critically impact cell function and lineage development. Particularly in male germ cells, mitochondria are uniquely clustered into intermitochondrial cement (IMC), an electron-dense granule in the cytoplasm to support proper spermatogenesis. But it remains puzzling how mitochondria assemble into such a stable structure as IMC without limiting membrane during development. Here, we showed that GASZ (germ cell-specific, ankyrin repeat, SAM and basic leucine zipper domain containing protein), a mitochondrion-localized germ cell-specific protein, self-interacted with each other to cluster mitochondria and maintain protein stability for IMC assembling. When the self-interaction of GASZ was disrupted by either deleting its critical interaction motif or using a blocking peptide, the IMC structure was destabilized, which in turn led to impaired spermatogenesis. Notably, the blocked spermatogenesis was reversible once GASZ self-interaction was recovered. Our findings thus reveal a critical mechanism by which mitochondrion-based granules are properly assembled to support germ cell development while providing an alternative strategy for developing nonhormonal male contraceptives by targeting IMC protein interactions.
PubMed: 38205030
DOI: 10.1093/pnasnexus/pgad480 -
BMC Biology Jan 2024Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders....
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.
Topics: Animals; Ankyrin Repeat; Catalytic Domain; Cell Nucleus; Drosophila; Histone Deacetylases; MEF2 Transcription Factors; Morphogenesis; Neurons
PubMed: 38167120
DOI: 10.1186/s12915-023-01800-1 -
Nature Communications Dec 2023Brain endothelial LDL receptor-related protein 1 (LRP1) is involved in the clearance of Aβ peptides across the blood-brain barrier (BBB). Here we show that endothelial...
Brain endothelial LDL receptor-related protein 1 (LRP1) is involved in the clearance of Aβ peptides across the blood-brain barrier (BBB). Here we show that endothelial deficiency of ankyrin repeat and SAM domain containing 1 A (ANKS1A) reduces both the cell surface levels of LRP1 and the Aβ clearance across the BBB. Association of ANKS1A with the NPXY motifs of LRP1 facilitates the transport of LRP1 from the endoplasmic reticulum toward the cell surface. ANKS1A deficiency in an Alzheimer's disease mouse model results in exacerbated Aβ pathology followed by cognitive impairments. These deficits are reversible by gene therapy with brain endothelial-specific ANKS1A. In addition, human induced pluripotent stem cell-derived BBBs (iBBBs) were generated from endothelial cells lacking ANKS1A or carrying the rs6930932 variant. Those iBBBs exhibit both reduced cell surface LRP1 and impaired Aβ clearance. Thus, our findings demonstrate that ANKS1A regulates LRP1-mediated Aβ clearance across the BBB.
Topics: Animals; Humans; Mice; Amyloid beta-Peptides; Blood-Brain Barrier; Brain; Endothelial Cells; Induced Pluripotent Stem Cells; Low Density Lipoprotein Receptor-Related Protein-1; Receptors, LDL
PubMed: 38123547
DOI: 10.1038/s41467-023-44319-3 -
Iranian Journal of Pharmaceutical... 2023Fibroblast growth factor 21 (FGF21) is a metabolic, endocrine hormone regulating insulin sensitivity, energy expenditure, and lipid metabolism. It has significant...
BACKGROUND
Fibroblast growth factor 21 (FGF21) is a metabolic, endocrine hormone regulating insulin sensitivity, energy expenditure, and lipid metabolism. It has significant potential as a therapeutic drug for treating type 2 diabetes and obesity. However, the clinical efficacy of FGF21 analogs is limited due to their instability and short half-life. Glucagon-like peptide 1 (GLP-1) receptor agonists have been recognized as effective medications for type 2 diabetes mellitus and obesity over the past two decades.
METHODS
This study designed a new long-acting dual-agonist, exendin-4/FGF21, utilizing albumin-binding-designed ankyrin repeat proteins (DARPins) as carriers. The purified fusion proteins were subcutaneously injected into mice for pharmacokinetic and biological activity studies.
RESULTS
Ex-DARP-FGF21 had a high binding affinity for human serum albumin (HSA) in vitro and a prolonged half-life of 27.6 hours in vivo. Bioactivity results reveal that Ex-DARP-FGF21 significantly reduced blood glucose levels in healthy mice. Moreover, compared to Ex-DARP alone, the Ex-DARP-FGF21 dual agonist displayed enhanced blood glucose lowering bioactivity and superior body weight management in the diet-induced obesity (DIO) mouse model.
CONCLUSIONS
These results indicate that the long-acting dual agonist of exendin-4 and FGF21 holds considerable potential as a treatment for type 2 diabetes mellitus (T2DM) and obesity in the future.
PubMed: 38116563
DOI: 10.5812/ijpr-131015 -
Nature Communications Dec 2023In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We...
In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.
Topics: Botulinum Toxins; Designed Ankyrin Repeat Proteins; Botulinum Toxins, Type A; Clostridium botulinum
PubMed: 38110403
DOI: 10.1038/s41467-023-44102-4 -
BMC Cancer Dec 2023The aim of this study is to evaluate an AAV vector that can selectively target breast cancer cells and to investigate its specificity and anti-tumor effects on breast...
OBJECTIVE
The aim of this study is to evaluate an AAV vector that can selectively target breast cancer cells and to investigate its specificity and anti-tumor effects on breast cancer cells both in vitro and in vivo, offering a new therapeutic approach for the treatment of EpCAM-positive breast cancer.
METHODS
In this study, a modified AAV2 viral vector was used, in which EpCAM-specific DARPin EC1 was fused to the VP2 protein of AAV2, creating a viral vector that can target breast cancer cells. The targeting ability and anti-tumor effects of this viral vector were evaluated through in vitro and in vivo experiments.
RESULTS
The experimental results showed that the AAV2M virus could specifically infect EpCAM-positive breast cancer cells and accurately deliver the suicide gene HSV-TK to tumor tissue in mice, significantly inhibiting tumor growth. Compared to the traditional AAV2 viral vector, the AAV2M virus exhibited reduced accumulation in liver tissue and had no impact on tumor growth.
CONCLUSION
This study demonstrates that AAV2M is a gene delivery vector capable of targeting breast cancer cells and achieving selective targeting in mice. The findings offer a potential gene delivery system and strategies for gene therapy targeting EpCAM-positive breast cancer and other tumor types.
Topics: Humans; Mice; Animals; Female; Epithelial Cell Adhesion Molecule; Designed Ankyrin Repeat Proteins; Breast Neoplasms; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Dependovirus
PubMed: 38082377
DOI: 10.1186/s12885-023-11705-5 -
Chinese Journal of Traumatology =... Jan 2024To identify the potential target genes of blast lung injury (BLI) for the diagnosis and treatment.
PURPOSE
To identify the potential target genes of blast lung injury (BLI) for the diagnosis and treatment.
METHODS
This is an experimental study. The BLI models in rats and goats were established by conducting a fuel-air explosive power test in an unobstructed environment, which was subsequently validated through hematoxylin-eosin staining. Transcriptome sequencing was performed on lung tissues from both goats and rats. Differentially expressed genes were identified using the criteria of q ≤ 0.05 and |log fold change| ≥ 1. Following that, enrichment analyses were conducted for gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathways. The potential target genes were further confirmed through quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay.
RESULTS
Observations through microscopy unveiled the presence of reddish edema fluid, erythrocytes, and instances of focal or patchy bleeding within the alveolar cavity. Transcriptome sequencing analysis identified a total of 83 differentially expressed genes in both rats and goats. Notably, 49 genes exhibited a consistent expression pattern, with 38 genes displaying up-regulation and 11 genes demonstrating down-regulation. Enrichment analysis highlighted the potential involvement of the interleukin-17 signaling pathway and vascular smooth muscle contraction pathway in the underlying mechanism of BLI. Furthermore, the experimental findings in both goats and rats demonstrated a strong association between BLI and several key genes, including anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4, which exhibited up-regulation.
CONCLUSIONS
Anterior gradient 2, ankyrin repeat domain 65, bactericidal/permeability-increasing fold containing family A member 1, bactericidal/permeability-increasing fold containing family B member 1, and keratin 4 hold potential as target genes for the prognosis, diagnosis, and treatment of BLI.
Topics: Rats; Animals; Lung Injury; Goats; Keratin-4; Gene Expression Profiling; Gene Expression
PubMed: 38071167
DOI: 10.1016/j.cjtee.2023.11.005