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Molecular Autism Apr 2024SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) monogenic mutations or deficiency leads to excessive stereotypic behavior and impaired sociability, which...
BACKGROUND
SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) monogenic mutations or deficiency leads to excessive stereotypic behavior and impaired sociability, which frequently occur in autism cases. To date, the underlying mechanisms by which Shank3 mutation or deletion causes autism and the part of the brain in which Shank3 mutation leads to the autistic phenotypes are understudied. The hypothalamus is associated with stereotypic behavior and sociability. p38α, a mediator of inflammatory responses in the brain, has been postulated as a potential gene for certain cases of autism occurrence. However, it is unclear whether hypothalamus and p38α are involved in the development of autism caused by Shank3 mutations or deficiency.
METHODS
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and immunoblotting were used to assess alternated signaling pathways in the hypothalamus of Shank3 knockout (Shank3) mice. Home-Cage real-time monitoring test was performed to record stereotypic behavior and three-chamber test was used to monitor the sociability of mice. Adeno-associated viruses 9 (AAV9) were used to express p38α in the arcuate nucleus (ARC) or agouti-related peptide (AgRP) neurons. D176A and F327S mutations expressed constitutively active p38α. T180A and Y182F mutations expressed inactive p38α.
RESULTS
We found that Shank3 controls stereotypic behavior and sociability by regulating p38α activity in AgRP neurons. Phosphorylated p38 level in hypothalamus is significantly enhanced in Shank3 mice. Consistently, overexpression of p38α in ARC or AgRP neurons elicits excessive stereotypic behavior and impairs sociability in wild-type (WT) mice. Notably, activated p38α in AgRP neurons increases stereotypic behavior and impairs sociability. Conversely, inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3 mice. In contrast, activated p38α in pro-opiomelanocortin (POMC) neurons does not affect stereotypic behavior and sociability in mice.
LIMITATIONS
We demonstrated that SHANK3 regulates the phosphorylated p38 level in the hypothalamus and inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3 mice. However, we did not clarify the biochemical mechanism of SHANK3 inhibiting p38α in AgRP neurons.
CONCLUSIONS
These results demonstrate that the Shank3 deficiency caused autistic-like behaviors by activating p38α signaling in AgRP neurons, suggesting that p38α signaling in AgRP neurons is a potential therapeutic target for Shank3 mutant-related autism.
Topics: Animals; Mice; Agouti-Related Protein; Arcuate Nucleus of Hypothalamus; Autistic Disorder; Hypothalamus; Microfilament Proteins; Nerve Tissue Proteins; Neurons; Mitogen-Activated Protein Kinase 14
PubMed: 38570876
DOI: 10.1186/s13229-024-00595-4 -
BioRxiv : the Preprint Server For... Jun 2024grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which...
grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify proteins that stimulate MAPK activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on high osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain.
PubMed: 38562771
DOI: 10.1101/2024.03.17.585421 -
Molecular Pharmaceutics Apr 2024HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such...
HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three Tc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (GC), (Gly-Gly-Gly-Ser)-Cys ((GS)C), or Glu-Glu-Glu-Cys (EC) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with Tc(CO) using the (HE)-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of Tc-labeled DARPin G3 variants to HER2 in vitro; however, [Tc]Tc-G3-(GS)C-HYNIC had the highest affinity. Comparative biodistribution of [Tc]Tc-G3-GC-HYNIC, [Tc]Tc-G3-(GS)C-HYNIC, [Tc]Tc-G3-EC-HYNIC, and [Tc]Tc-(HE)-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [Tc]Tc-G3-EC-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [Tc]Tc-G3-(GS)C-HYNIC and [Tc]Tc-(HE)-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [Tc]Tc-G3-(GS)C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [Tc]Tc-(HE)-G3. Radiolabeling of DARPin G3-HYNIC conjugates with Tc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the Tc-tricarbonyl-labeled DARPin G3. At this stage, [Tc]Tc-(HE)-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Designed Ankyrin Repeat Proteins; Molecular Imaging; Neoplasms; Tissue Distribution; Receptor, ErbB-2
PubMed: 38557163
DOI: 10.1021/acs.molpharmaceut.3c01173 -
Protein Science : a Publication of the... Apr 2024Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on...
Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.
Topics: Designed Ankyrin Repeat Proteins; Proteins
PubMed: 38533546
DOI: 10.1002/pro.4973 -
Autophagy Jul 2024A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the...
A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the molecular and neuronal mechanisms are not fully understood. Here, we reported that impaired neuronal macroautophagy in the prelimbic cortical (PrL) subregion of the mPFC paralleled the occurrence of anxiety-like behaviors in rats with chronic spared nerve injury (SNI). Intriguingly, such macroautophagy impairment was mainly observed in a FOS/c-Fos neuronal subpopulation in the PrL. Chemogenetic inactivation of this comorbid anxiety-related neuronal ensemble relieved pain-induced anxiety-like behaviors. Rescuing macroautophagy impairment in this neuronal ensemble relieved chronic pain-associated anxiety and mechanical allodynia and restored synaptic homeostasis at the molecular level. By contrast, artificial disruption of macroautophagy induced early-onset co-morbid anxiety in neuropathic rats, but not general anxiety in normal rats. Taken together, our work identifies causal linkage between PrL neuronal macroautophagy dysfunction and comorbid anxiety in neuropathic pain and provides novel insights into the role of PrL by differentiating its contribution in pain-induced comorbid anxiety from its modulation over general anxiety-like behaviors. AAV: adeno-associated viruses; ACC: anterior cingulate cortex; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CNO: clozapine--oxide; CQ: chloroquine; DIA: data independent acquisition; DIO: double floxed inverse orf; DLG4/PSD-95: discs large MAGUK scaffold protein 4; Dox: doxycycline; GABA: γ-aminobutyric acid; GFP: green fluorescent protein; GO: gene ontology; Gi: inhibitory guanine nucleotide-binding proteins; HsCHRM4/M4D: human cholinergic receptor muscarinic 4; HsSYN: human synapsin; KEGG: Kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; LC3-II: PE conjugated microtubule-associated protein 1 light chain3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mPFC: medial prefrontal cortex; P2A: 2A self-cleaving peptide; PPI: protein-protein interaction networks; PrL: prelimbic cortex; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; rtTA: reverse tetracycline-transactivator; SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; SHANK3: SH3 and multiple ankyrin repeat domains 3; SLC1A1/EAAC1: solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, systemXag), member 1; SNAP23: synaptosomal-associated protein 23; SNI:spared nerve injury; SQSTM1/p62: sequestosome 1; SYT3: synaptotagmin 3; TRE: tetracycline-responsive element; TRE3G: third-generation tetracycline-responsive element.
Topics: Animals; Neuralgia; Prefrontal Cortex; Anxiety; Rats; Neurons; Male; Macroautophagy; Rats, Sprague-Dawley; Behavior, Animal; Chronic Pain; Autophagy; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Hyperalgesia
PubMed: 38522078
DOI: 10.1080/15548627.2024.2330038 -
Heliyon Mar 2024KBG syndrome is a rare autosomal dominant condition characterized by multisystem developmental disorder, primarily caused by loss-of-function variants in ankyrin repeat...
KBG syndrome is a rare autosomal dominant condition characterized by multisystem developmental disorder, primarily caused by loss-of-function variants in ankyrin repeat domain-containing protein 11 (ANKRD11). Approximately 80 % of variants associated with KBG syndrome, are frameshift and nonsense variants. Current insight into the pathogenesis of KBG syndrome resulting from truncating variants remains limited. Here, we presented two members from a non-consanguineous Chinese pedigree both exhibiting characteristics fitting the KBG syndrome-associated phenotypic spectrum. Whole-exome sequencing identified a novel heterozygous frameshift variant in ANKRD11 (NM_013275.6, c.2280_2281delGT, p.Y761Qfs*20) in the proband. Sanger sequencing confirmed that the variant was inherited from her mother and co-segregated with KBG syndrome phenotype. functional assays revealed that the frameshift variant escaped nonsense-mediated mRNA decay, and resulting in a truncated protein with significantly increased expression levels compared to full-length ANKRD11. Immunofluorescence results demonstrated that truncated protein was predominantly expressed in the nucleus of HEK293 cells, while wild-type ANKRD11 was equally distributed in both the nucleus and cytoplasm. Moreover, the truncated protein significantly reduced /-promoter luciferase activity in comparison to wild-type ANKRD11 protein, as well as a remarkably decrease in the endogenous 1 mRNA level in HEK293 cells. These findings suggest a loss of transcriptional activation function and potentially a dominant-negative mechanism. Overall, our study expands the mutational spectrum of gene and provides new insights into the pathogenic mechanism of KBG syndrome caused by truncating variants.
PubMed: 38515699
DOI: 10.1016/j.heliyon.2024.e28082 -
Protein Science : a Publication of the... Apr 2024Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors...
Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N-terminal RBP-j-associated molecule (RAM) region and ankyrin repeat (ANK) domain bind transcription factor CSL and recruit co-activator Mastermind-like-1 (MAML1) to activate transcription. However, different paralogs can lead to distinct outcomes. To better understand paralog-specific differences in Notch signaling, we performed a thermodynamic analysis of the Notch transcriptional activation complexes for all four Notch paralogs using isothermal titration calorimetry. Using chimeric constructs, we find that the RAM region is the primary determinant of stability of binary RAMANK:CSL complexes, and that the ANK regions are largely the determinants of MAML1 binding to pre-formed RAMANK:CSL complexes. Free energies of these binding reactions (ΔG and ΔG) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔG, where ΔG = ΔG + ΔG). To probe how these affinity differences affect Notch signaling, we performed transcriptional activation assays with the paralogous and chimeric NICDs, and analyzed the results with an independent multiplicative model that quantifies contributions of the paralogous RAM, ANK, and C-terminal regions (CTR) to activation. This analysis shows that transcription activation correlates with ΔG, but that activation is further modified by CTR identity in a paralog-specific way.
Topics: Humans; Transcriptional Activation; Receptors, Notch; Protein Binding; Gene Expression Regulation; Thermodynamics; DNA-Binding Proteins; Transcription Factors
PubMed: 38511488
DOI: 10.1002/pro.4947 -
Biochemistry Apr 2024AQP4-IgG is an autoantibody associated with neuromyelitis optica spectroscopic disorder (NMOSD), a central nervous system inflammatory disease that requires early...
AQP4-IgG is an autoantibody associated with neuromyelitis optica spectroscopic disorder (NMOSD), a central nervous system inflammatory disease that requires early diagnosis and treatment. We designed two fusion proteins, AQP4-DARPin1 and AQP4-DARPin2, comprising the complete antigenic epitopes of aquaporin-4 (AQP4) and the constant region of the scaffold protein DARPin. These fusion proteins were expressed and purified from and coated on microplates to develop an efficient method for detecting AQP4-IgG. Molecular dynamics simulation revealed that the fusion of AQP4 extracellular epitopes with DARPin did not alter the main structure of DARPin. The purified AQP4-DARPins bound recombinant antibody rAb-53 (AQP4-IgG) with affinities of 135 and 285 nM, respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation demonstrated that AQP4-DARPin1 specifically recognized AQP4-IgG in the NMOSD patient serum. AQP4-DARPin1 as a coated antigen showed higher ELISA signal and end point dilution ratio than full-length AQP4. Our AQP4-DARPin1-coated AQP4-IgG ELISA had 100% specificity and 90% sensitivity. These results indicate that AQP4-DARPin1, compared to existing detection strategies that use full-length or extracellular loop peptides of AQP4, provides a new and more effective approach to the ELISA detection of NMOSD.
Topics: Humans; Neuromyelitis Optica; Designed Ankyrin Repeat Proteins; Aquaporin 4; Epitopes; Immunoglobulin G
PubMed: 38498694
DOI: 10.1021/acs.biochem.3c00688 -
ACS Chemical Biology Apr 2024Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in...
Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in trans on the cell surface of the effector and target cell to induce proximal immune effects, opening exciting windows for immunotherapies. To date, the engineering of BsAbs has been mainly focused on tuning the molecular weight and valency. However, the effects of spatial factors on the biological functions of BsAbs have been less explored due to the lack of biochemical methods to precisely manipulate protein geometry. Here, we studied the geometric effects of the TcEs. First, by genetically inserting rigidly designed ankyrin repeat proteins into TcEs, we revealed that the efficacy progressively decreased as the spacer distance of the two binding domains increased. Then, we constructed 26 pairs of TcEs with the same size but varying orientations using click chemistry-mediated conjugation at different mutation sites. We found that linear ligation sites play a minor role in modulating cell-killing efficacy. Next, we rendered the TcEs' advanced topology by cyclization chemistry using the SpyTag/SpyCatcher pair or sortase ligation approaches. Cyclized TcEs were generally more potent than their linear counterparts. Particularly, sortase A cyclized TcEs, bearing a minimal tagging motif, exhibited better cell-killing efficacy in vitro and improved stability both in vitro and in vivo compared to the linear TcE. This work combines modern bioconjugation chemistry and protein engineering tools for antibody engineering, shedding light on the elusive spatial factors of BsAbs functionality.
Topics: Antibodies, Bispecific; Click Chemistry; Protein Engineering; Proteins; T-Lymphocytes; Humans
PubMed: 38491942
DOI: 10.1021/acschembio.3c00728 -
ACS Nano Mar 2024The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and...
The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.
Topics: Actins; Designed Ankyrin Repeat Proteins; Cytoskeleton; Actin Cytoskeleton; Microfilament Proteins
PubMed: 38489155
DOI: 10.1021/acsnano.3c12265