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Acta Neuropathologica Communications Jul 2023Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis...
Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1β, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.
Topics: Animals; Mice; Antibodies, Monoclonal; Epitopes; Immunization; Intermediate Filaments; NF-kappa B
PubMed: 37434215
DOI: 10.1186/s40478-023-01592-z -
MBio Aug 2023HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK...
HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.
Topics: Humans; Down-Regulation; HIV-1; Ligands; Antibody-Dependent Cell Cytotoxicity; Killer Cells, Natural; Signaling Lymphocytic Activation Molecule Family; HIV Infections
PubMed: 37404017
DOI: 10.1128/mbio.00789-23 -
Frontiers in Cell and Developmental... 2023PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common...
PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common catalytic (C-subunit encoded by PPP2CA/PPP2CB), and one of many variable regulatory (B) subunits. The site of phosphoprotein phosphatase (PPP) hydrolysis features a bimetal system (M/M), an associated bridge hydroxide [W(OH)], and a highly-conserved core sequence. In the presumptive common mechanism, the phosphoprotein's seryl/threonyl phosphate coordinates the M/M system, W(OH) attacks the central P atom, rupturing the antipodal bond, and simultaneously, a histidine/aspartate tandem protonates the exiting seryl/threonyl alkoxide. Based on studies of PPP5C, a conserved arginine proximal to M is also expected to bind the substrate's phosphate group in a bidentate fashion. However, in PP2A isozymes, the role of the arginine (Arg) in hydrolysis is not clear because two independent structures for PP2A(PPP2R5C) and PP2A(PPP2R5D) show that Arg engages in a weak salt bridge at the B:C interface. These observations raise the question of whether hydrolysis proceeds with or without direct involvement of Arg. The interaction of Arg with B:Glu in PP2A(PPP2R5D) is significant because the pathogenic E198K variant of B56δ is associated with irregular protein phosphorylation levels and consequent developmental disorders (Jordan's Syndrome; OMIM #616355). In this study, we perform quantum-based hybrid [ONIOM(UB3LYP/6-31G(d):UPM7)] calculations on 39-residue models of the PP2A(PPP2R5D)/pSer (phosphoserine) system to estimate activation barriers for hydrolysis in the presence of bidentate Arg-substrate binding and when Arg is otherwise engaged in the salt-bridge interaction. Our solvation-corrected results yield ΔH ≈ ΔE = +15.5 kcal/mol for the former case, versus +18.8 kcal/mol for the latter, indicating that bidentate Arg-substrate binding is critical for optimal catalytic function of the enzyme. We speculate that PP2A(PPP2R5D) activity is suppressed by B:Glu sequestration of C:Arg under native conditions, whereas the PP2A(PPP2R5D)-holoenzyme containing the E198K variant has a positively-charged lysine in this position that alters normal function.
PubMed: 37377738
DOI: 10.3389/fcell.2023.1141804 -
Molecules (Basel, Switzerland) Jun 2023In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity...
In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.
Topics: Phosphorylation; src Homology Domains; Proto-Oncogene Proteins pp60(c-src); Phosphoserine; Serine
PubMed: 37375241
DOI: 10.3390/molecules28124686 -
Nature Communications Jun 2023Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains...
Altered protein phosphorylation in cancer cells often leads to surface presentation of phosphopeptide neoantigens. However, their role in cancer immunogenicity remains unclear. Here we describe a mechanism by which an HLA-B*0702-specific acute myeloid leukemia phosphoneoantigen, pMLL (EPR(pS)PSHSM), is recognized by a cognate T cell receptor named TCR27, a candidate for cancer immunotherapy. We show that the replacement of phosphoserine P with serine or phosphomimetics does not affect pMHC conformation or peptide-MHC affinity but abrogates TCR27-dependent T cell activation and weakens binding between TCR27 and pMHC. Here we describe the crystal structures for TCR27 and cognate pMHC, map of the interface produced by nuclear magnetic resonance, and a ternary complex generated using information-driven protein docking. Our data show that non-covalent interactions between the epitope phosphate group and TCR27 are crucial for TCR specificity. This study supports development of new treatment options for cancer patients through target expansion and TCR optimization.
Topics: Humans; Phosphopeptides; Receptors, Antigen, T-Cell; Protein Binding
PubMed: 37353482
DOI: 10.1038/s41467-023-39425-1 -
The Plant Cell Sep 2023Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether...
Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.
Topics: Catalase; Oryza; Hydrogen Peroxide; Protein Phosphatase 1; Salt Tolerance; Homeostasis; Plant Proteins
PubMed: 37325884
DOI: 10.1093/plcell/koad167 -
The Journal of Biological Chemistry Jul 2023Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis....
Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1β. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1β induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1β-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1β-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.
Topics: Animals; Humans; Mice; Atherosclerosis; Cells, Cultured; Hyperplasia; Inflammation; Interleukin-1 Receptor-Associated Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Phosphoserine; TNF Receptor-Associated Factor 6; Ubiquitin Thiolesterase; NF-kappa B; Carotid Arteries; Interleukin-1beta; Ubiquitination
PubMed: 37311534
DOI: 10.1016/j.jbc.2023.104911 -
Cureus May 2023Neu-Laxova syndrome (NLS) is a rare lethal disorder with autosomal recessive inheritance and is characterized by multiple congenital anomalies. Our case of NLS presented...
Neu-Laxova syndrome (NLS) is a rare lethal disorder with autosomal recessive inheritance and is characterized by multiple congenital anomalies. Our case of NLS presented with severe intrauterine growth restriction (IUGR), abnormal facial features, severe central nervous system malformations, skeletal muscle contractures, and the hallmark signs of NLS: ichthyotic skin and excessive subcutaneous tissue with edema. Additionally, testing amniotic fluid from a prior pregnancy with a fetus showing similar abnormalities revealed several regions of homozygosity; one of these regions involved chromosome 1p13.2-p11.2, where the gene is located. Based on the pattern of findings on serial fetal ultrasounds, postmortem neonatal exams, gross and microscopic exams, radiographs, and genetic analysis in conjunction with the clinical history and the prior pregnancy with the above-described molecular alteration, a final diagnosis of NLS was made. This rare developmental disorder is characterized by heterogenous neuroectodermal defects. Fetal ultrasound in the second trimester can help diagnose it. It is postulated to be caused by loss-of-function mutations in the (phosphoglycerate dehydrogenase), (phosphoserine aminotransferase 1), and (phosphoserine phosphatase) genes, which are responsible for de novo L-serine synthesis.
PubMed: 37303350
DOI: 10.7759/cureus.38787 -
Regenerative Biomaterials 2023Flexible hydrogels containing various osteogenic inorganic constituents, which can accommodate complicated shape variations, are considered as ideal grafts for...
Flexible hydrogels containing various osteogenic inorganic constituents, which can accommodate complicated shape variations, are considered as ideal grafts for craniofacial bone defect reconstruction. However, in most hybrid hydrogels, poor interaction between the polymer network and particles has detrimental effects on hydrogel rheological and structural properties, clinical manipulation and repair efficacy. In this article, we designed and prepared a series of hyaluronic acid composite hydrogel containing Cu-doped bioactive glass (CuBG) and phosphoserine (PS), in which hyaluronic acid was modified by methacrylate groups and phenylboronic acid groups to form a double crosslinked network. PS acted as an interaction bridge of CuBG particles and HAMA-PBA network to improve the mechanical properties of the composite hydrogels. The CuBG/PS hydrogels exhibited suitable rheological properties (injectable, self-healing, shape-adaptable), bone tissue integrating ability and anti-bacterial property. Meanwhile, we found that CuBG and PS have synergistic effect on improving osteogenic efficiency both and , particularly when the ratio of CuBG to PS is lower than 3 (9CB/3PS). This work provided a versatile and scalable approach to enhanced the interaction within inorganic particles and polymer network in hydrogels without extra modification on components.
PubMed: 37287897
DOI: 10.1093/rb/rbad054