Phosphorylation Of Residues Research Articles

Marmosets as model systems for the study of Alzheimer's disease and related dementias: Substantiation of physiological tau 3R and 4R isoform expression and phosphorylation.

Marmosets spontaneously develop pathological hallmarks of Alzheimer's disease (AD) including amyloid beta plaques. However, tau expression in the marmoset brain has been understudied. Isoforms of tau were examined by western blot, mass spectrometry, immunofluorescence, and immunohistochemical staining. 3R and 4R tau isoforms are expressed in marmoset brains at both the transcript and protein levels across ages. Mass spectrometry analysis revealed that tau peptides in marmoset corresponded to the 3R and 4R peptides in human brain, with 3R predominating at birth and an ≈40%:60% 3R:4R ratios in adolescents and adults; tau was distributed widely in neurons, with localization in the soma and synaptic regions. Phosphorylation residues were observed on Threonine (Thr) Thr181, Thr217, Thr231, Serine (Ser) Ser202/Thr205, and Ser396/Ser404. Our results confirm both 3R and 4R tau isoform expression and phosphorylation residues in the marmoset brain, and emphasize the significance of marmosets with natural expression of AD-related hallmarks as important translational models for AD. Highlights We report comprehensive characterization of tau isoform expression in marmoset brains across the lifespan. 3R and 4R tau isoforms are expressed in marmoset brains at both the transcript and protein levels across ages. These data emphasize the significance of marmosets with natural expression of primate-specific traits that are important for the study of Alzheimer's disease.

CSIG-31. ANNEXIN A7 ALTERNATIVE SPLICING IN GLIOMA STEM CELLS FACILITATES THE DEGRADATION OF EGFR BY PHOSPHORYLATING TYROSINE RESIDUES EXCLUSIVE TO PROTEIN ISOFORM 1

Abstract The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling and resulting in hyperactive RTK signaling. Exon six of Annexin A7 (ANXA7) can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates in PDX glioma stem cells (GSCs). We demonstrated that ANXA7 exon skipping supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. JX14 GSCs were made to overexpress ANXA7-I1 with lentivirus to conduct immunofluorescence experiments measuring EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway for the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (R2=0.9267, P<0.0001). Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). On the other hand, ANXA7 siRNA KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1 to prefer recycling again while EV cells were unaffected. I1 overexpression significantly reduced EGFR signaling through degradation, which is driven by the phosphorylation of I1-exclusive tyrosine residues in phosphomimetic (p<0.01) and non-phosphorylatable protein mutants. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for GSCs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition.

GPS-pPLM: A Language Model for Prediction of Prokaryotic Phosphorylation Sites.

In the prokaryotic kingdom, protein phosphorylation serves as one of the most important posttranslational modifications (PTMs) and is involved in orchestrating a broad spectrum of biological processes. Here, we report an updated online server named the group-based prediction system for prokaryotic phosphorylation language model (GPS-pPLM), used for predicting phosphorylation sites (p-sites) in prokaryotes. For model training, two deep learning methods, a transformer and a deep neural network, were employed, and a total of 10 sequence features and contextual features were integrated. Using 44,839 nonredundant p-sites in 16,041 proteins from 95 prokaryotes, two general models for the prediction of O-phosphorylation and N-phosphorylation were first pretrained and then fine-tuned to construct 6 predictors specific for each phosphorylatable residue type as well as 134 species-specific predictors. Compared with other existing tools, the GPS-pPLM exhibits higher accuracy in predicting prokaryotic O-phosphorylation p-sites. Protein sequences in FASTA format or UniProt accession numbers can be submitted by users, and the predicted results are displayed in tabular form. In addition, we annotate the predicted p-sites with knowledge from 22 public resources, including experimental evidence, 3D structures, and disorder tendencies. The online service of the GPS-pPLM is freely accessible for academic research.

A single phosphorylatable amino acid residue is essential forthe recognition of multiple potyviral HCPro effectors by potato Nytbr.

Potato virus Y (PVY, Potyviridae) is among the most important viral pathogens of potato. The potato resistance gene Nytbr confers hypersensitive resistance to the ordinary strain of PVY (PVYO), but not the necrotic strain (PVYN). Here, we unveil that residue 247 of PVY helper component proteinase (HCPro) acts as a central player controlling Nytbr strain-specific activation. We found that substituting the serine at 247 in the HCPro of PVYO (HCProO) with an alanine as in PVYN HCPro (HCProN) disrupts Nytbr recognition. Conversely, an HCProN mutant carrying a serine at position 247 triggers defence. Moreover, we demonstrate that plant defences are induced against HCProO mutants with a phosphomimetic or another phosphorylatable residue at 247, but not with a phosphoablative residue, suggesting that phosphorylation could modulate Nytbr resistance. Extending beyond PVY, we establish that the same response elicited by the PVYO HCPro is also induced by HCPro proteins from other members of the Potyviridae family that have a serine at position 247, but not by those with an alanine. Together, our results provide further insights in the strain-specific PVY resistance in potato and infer a broad-spectrum detection mechanism of plant potyvirus effectors contingent on a single amino acid residue.

CABS1 Is Essential for Progressive Motility and the Integrity of Fibrous Sheath in Mouse Epididymal Spermatozoa.

The calcium-binding protein spermatid-associated 1 (CABS1) localizes to the principal piece of mature sperm flagella. Deletion of CABS1 results in subfertility in male mice, possibly due to an impaired annulus in the sperm flagella. However, it is unknown whether there are other mechanisms by which CABS1 affects male fertility. Our current investigation has uncovered that CABS1 is located in the midsection of the flagellum in testicular sperm and the principal piece in epididymal sperm. Moreover, male mice lacking CABS1 exhibit a defect in the progressive motility of sperm. Furthermore, the regulation of calcium levels, which has been reported to have a significant impact on sperm motility, capacitation, and the acrosome reaction, is also affected when sperm are exposed to alkalized high-salt buffer (pH 8.0) and progesterone (100 μM) in Cabs1-null spermatozoa. This alteration in calcium response may contribute to changes in the phosphorylation of PKA substrates and subsequent phosphorylation of tyrosine residues. Additionally, the absence of CABS1 leads to a defective fibrous sheath and abnormal configuration of doublet microtubules in post-testicular sperm. These findings indicate that the absence of CABS1 also disrupts the structural integrity of the fibrous sheath, resulting in male subfertility. The highly conserved nature of CABS1 in humans suggests that it could potentially be a contributing factor to asthenozoospermia in men.

Phosphorylation-driven epichaperome assembly is a regulator of cellular adaptability and proliferation

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90’s interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine.

Phosphorylation of Ser711 residue in the hypervariable region of zoonotic genotype 3 hepatitis E virus is important for virus replication.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

Ligand requirements for immunoreceptor triggering

Leukocytes interact with other cells using cell surface receptors. The largest group of such receptors are non-catalytic tyrosine phosphorylated receptors (NTRs), also called immunoreceptors. NTR signalling requires phosphorylation of cytoplasmic tyrosine residues by SRC-family tyrosine kinases. How ligand binding to NTRs induces this phosphorylation, also called NTR triggering, remains controversial, with roles suggested for size-based segregation, clustering, and mechanical force. Here we exploit a recently developed cell-surface generic ligand system to explore the ligand requirements for NTR triggering. We examine the effect of varying the ligand’s length, mobility and valency on the activation of representative members of four NTR families: SIRPβ1, Siglec 14, NKp44 and TREM-1. Increasing the ligand length impairs activation via NTRs, despite enhancing cell-cell conjugation, while varying ligand mobility has little effect on either conjugation or activation. Increasing the valency of the ligand, while enhancing cell-cell conjugation, does not enhance activation at equivalent levels of conjugation. These findings are more consistent with a role for size-based segregation, rather than mechanical force or clustering, in NTR triggering, suggesting a role for the kinetic-segregation model.

The brassinosteroid receptor StBRI1 promotes tuber development by enhancing plasma membrane H+-ATPase activity in potato.

The brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) plays a critical role in plant growth and development. Although much is known about how BR signaling regulates growth and development in many crop species, the role of StBRI1 in regulating potato (Solanum tuberosum) tuber development is not well understood. To address this question, a series of comprehensive genetic and biochemical methods were applied in this investigation. It was determined that StBRI1 and Solanum tuberosum PLASMA MEMBRANE (PM) PROTON ATPASE2 (PHA2), a PM-localized proton ATPase, play important roles in potato tuber development. The individual overexpression of StBRI1 and PHA2 led to a 22% and 25% increase in tuber yield per plant, respectively. Consistent with the genetic evidence, in vivo interaction analysis using double transgenic lines and PM H+-ATPase activity assays indicated that StBRI1 interacts with the C-terminus of PHA2, which restrains the intramolecular interaction of the PHA2 C-terminus with the PHA2 central loop to attenuate autoinhibition of PM H+-ATPase activity, resulting in increased PHA2 activity. Furthermore, the extent of PM H+-ATPase autoinhibition involving phosphorylation-dependent mechanisms corresponds to phosphorylation of the penultimate Thr residue (Thr-951) in PHA2. These results suggest that StBRI1 phosphorylates PHA2 and enhances its activity, which subsequently promotes tuber development. Altogether, our results uncover a BR-StBRI1-PHA2 module that regulates tuber development and suggest a prospective strategy for improving tuberous crop growth and increasing yield via the cell surface-based BR signaling pathway.

The mTOR pathway controls phosphorylation of BRAF at T401

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

The multifaceted role of different SnRK gene family members in regulating multiple abiotic stresses in plants.

Abiotic stresses are a major constraint for agricultural productivity and food security in today's era of climate change. Plants can experience different types of abiotic stresses, either individually or in combination. Sometimes, more than one stress event may occur simultaneously or one after another during the lifecycle of the plant. In general, key survival strategies for stress tolerance may differ from one stress to another. However, at the molecular level, evolutionarily conserved protein kinase SUCROSE NONFERMENTING 1 (SNF1)-related protein kinase (SnRK) gene family members, comprising SnRK1, SnRK2, and SnRK3 gene families, play a key role in different types of stress and adaptive responses. SnRK gene family members can act as master regulators and regulate the central metabolism of plants, which determines the energy distribution in either survival or growth/developmental processes. The key mechanism of SnRK-mediated regulation is associated with the phosphorylation of downstream genes, which either induces or dampens the function of target proteins. This may be crucial for maintaining differential morpho-physiological and biochemical processes in plants, including potassium signalling, ROS homeostasis, sugar signalling, and energy homeostasis. Furthermore, phosphorylation sites associated with different targets were also reviewed, which showed that SnRK-mediated phosphorylation of Serine and Threonine residues of the target protein is a site-specific event, where the target consists of specific amino acid sequences, including RXXS/T, Serine-threonine rich regions, or AMPK/SNF1 types. Here, we review different classes of SnRK gene family members and their multifaceted roles in understanding the commonality of SnRK-mediated responses to multiple abiotic stresses in plants.

The outcome of haematopoietic stem cell transplantation in a patient with STAT1 gain-of-function: a case report

Background: The human signal transducer and activator of transcription 1 (STAT1) is a latent cytoplasmic transcription factor and one of seven members of the STAT family. Autosomal dominant (AD) STAT1 gain-of-function (GOF), characterized by enhanced phosphorylation of the tyrosine-701 residue and STAT1 activation, is commonly associated with chronic mucocutaneous candidiasis (CMCC), immunodeficiency, aneurysms, malignancies, and autoimmune phenomena. The best management strategies for patients with STAT1 GOF remain unclear. Typically, the standard care includes supportive treatments such as antimicrobial prophylaxis, either alone or combined with immunoglobulin replacement therapy. Biological therapies such as JAK inhibitors have been shown to alleviate symptoms of infection and autoimmune disorders in patients with STAT1 GOF mutations. However, there is a lack of long-term outcome data for JAK inhibition. Hematopoietic stem cell transplantation (HSCT) is an alternative treatment option for a subset who experience a persistent disease course despite conventional therapy. However, the effectiveness of HSCT for this condition is not yet well established. Methods: Our patient’s medical records were analyzed retrospectively, including her medical history. Results: We present the outcome of HSCT performed on a 27-year-old female with STAT1 GOF, conducted as a treatment for acute myeloid leukemia (AML). Conclusion: HSCT may serve as an alternative and potentially curative treatment for certain STAT1 GOF patients with progressive, life-threatening conditions that do not respond to conventional therapies. Additional research is needed to improve the management of these patients. Statement of Novelty: We present a novel case study of HSCT as a treatment for AML in a 27-year-old patient with STAT1 GOF, highlighting the potential for curative outcomes in progressive, life-threatening conditions unresponsive to conventional therapies. This case contributes to the limited body of evidence supporting the efficacy and challenges of HSCT in STAT1 GOF patients.

Hypoxia-induced LAMB2-enriched extracellular vesicles promote peritoneal metastasis in gastric cancer via the ROCK1-CAV1-Rab11 axis.

Peritoneal metastasis is one of the most common risk factors contributing to the poor prognosis of gastric cancer. We previously reported that extracellular vesicles from gastric cancer cells could facilitate peritoneal metastasis. However, their impact on gastric cancer-induced peritoneal metastasis under hypoxic conditions remains unclear. This study aims to elucidate how hypoxia-resistant gastric cancer cell-derived extracellular vesicles affect the peritoneal metastasis of normoxic gastric cancer cells. Proteomic analysis revealed elevated levels of Caveolin1 and Laminin β2 in hypoxia-resistant gastric cancer cells and their corresponding extracellular vesicles. Importantly, Caveolin1 was found to play a central role in mediating Laminin β2 sorting into extracellular vesicles derived from hypoxia-resistant gastric cancer cells, and subsequently, extracellular vesicle-associated Laminin β2 promoted peritoneal metastasis in normoxic gastric cancer cells by activating the AKT pathway. Further investigation confirmed that Caveolin1 activation by Rho-related Coiled-coil kinase 1-mediated phosphorylation of Y14 residue is a key factor facilitating Laminin β2 sorting into extracellular vesicles. Moreover, Y14 phosphorylated- Caveolin1 enhanced Laminin β2 sorting by activating Rab11. Finally, our study demonstrated that a combined assessment of plasma extracellular vesicle-associated Caveolin1 and extracellular vesicle-associated Laminin β2 could provide an accurate predictive tool for peritoneal metastasis occurrence in gastric cancer.

Phosphoryl group wires stabilize pathological tau fibrils as revealed by multiple quantum spin counting NMR.

Hyperphosphorylation of the protein tau is one of the biomarkers of neurodegenerative diseases in the category of tauopathies. However, the molecular level, mechanistic, role of this common post-translational modification (PTM) in enhancing or reducing the aggregation propensity of tau is unclear, especially considering that combinatorial phosphorylation of multiple sites can have complex, non-additive, effects on tau protein aggregation. Since tau proteins stack in register and parallel to elongate into pathological fibrils, phosphoryl groups from adjacent tau strands with 4.8 Å separation must find an energetically favorable spatial arrangement. At first glance, this appears to be an unfavorable configuration due to the proximity of negative charges between phosphate groups from adjacent neighboring tau fibrils. However, this study tests a counterhypothesis that phosphoryl groups within the fibril core-forming segments favorably assemble into highly ordered, hydrogen-bonded, one-dimensionally extended wires under biologically relevant conditions. We selected two phosphorylation sites associated with neurodegeneration, serine 305 (S305p) and tyrosine 310 (Y310p), on a model tau peptide jR2R3-P301L (tau295-313) spanning the R2/R3 splice junction of tau, that readily aggregate into a fibril with characteristics of a seed-competent mini prion. Using multiple quantum spin counting (MQ-SC) by 31P solid-state NMR of phosphorylated jR2R3-P301L tau peptide fibrils, enhanced by dynamic nuclear polarization, we find that at least six phosphorous spins must neatly arrange in 1D within fibrils or in 2D within a protofibril to yield the experimentally observed MQ-coherence orders of four. We found that S305p stabilizes the tau fibrils and leads to more seeding-competent fibrils compared to jR2R3 P301L or Y310p. This study introduces a new concept that phosphorylation of residues within a core forming tau segment can mechanically facilitate fibril registry and stability due a hitherto unrecognized role of phosphoryl groups to form highly ordered, extended, 1D wires that stabilize pathological tau fibrils.

Detection of Hydrogen Ions Resulting from the Inhibition of Acetylcholinesterase Using Indium Tin Oxide-Coated Nanostructures for Pesticide Sensing

The neurotransmitter acetylcholine (ACh) is hydrolyzed by acetylcholinesterase (AChE) into acetic acid and choline to terminate synaptic transmission (1). Organophosphorus (OPs) found in pesticides induce toxicity in organisms by irreversibly binding to AChE, leading to the phosphorylation of the serine residue at the active site. ACh cannot undergo hydrolysis by the phosphorylated enzyme, resulting in excessive stimulation of nerves and muscles, akin to a nerve agent (2). Our objective is to develop a rapid detection tool for determining the levels of pesticide residues in dairy products. In this study, we measured the release of hydrogen ions (H+) resulting from the hydrolysis of ACh by AChE, serving as an indirect method to evaluate the inhibitory effect of OPs on AChE. To detect the signals of H+ released during ACh decomposition, we used ITO-coated nanostructures modified with 3-aminopropyl trimethoxysilane (APTMS) as a sensing substrate integrated into the extended gate of a field-effect transistor (EGFET) biosensor. The amino group at the end of APTMS can bond with H+. By monitoring the changes in electrical signal from the EGFET biosensor, it is possible to know the concentration of H+ in the solution. The output characteristics (Id-Vd) of the APTMS-modified ITO-VASiNW EGFET were measured using ACh solutions at different concentrations. A correlation was observed between Id and ACh concentration within the tested range, exhibiting a sensitivity of 0.016 mA1/2 / mM and an R2 value of 0.9. A lower concentration of H+ indicates a more pronounced inhibitory effect on AChE by OPs and suggests higher levels of pesticide residues. References (1) Colović, M. B., Krstić, D. Z., Lazarević-Pašti, T. D., Bondžić, A. M., & Vasić, V. M. (2013). Acetylcholinesterase inhibitors: pharmacology and toxicology. Curr Neuropharmacol, 11(3), 315-335.(2) Rajagopalan, V., Venkataraman, S., Rajendran, D. S., Vinoth Kumar, V., Kumar, V. V., & Rangasamy, G. (2023). Acetylcholinesterase biosensors for electrochemical detection of neurotoxic pesticides and acetylcholine neurotransmitter: A literature review. Environmental Research, 227, 115724. Figure 1

Nemo-like kinase blocks myeloid differentiation by targeting tumor suppressor C/EBPα in AML.

CCAAT/enhancer-binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine-protein kinase NLK, also known as Nemo-like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin-mediated degradation. Individual phospho-point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML.

LcMPK3 and LcMPK6 positively regulate fruitlet abscission in litchi

Mitogen-activated protein kinase (MAPK) cascades have been discovered to play a fundamental role in regulating organ abscission. However, the identity of protein substrates targeted by MAPK cascades, as well as whether the role of MAPK protein cascades in the abscission process is conserved across different plant species, remain unknown. Here, the role of homologs of MPK3 and MPK6 in regulating fruit abscission were characterized in litchi. Ectopic expression of LcMPK3 or LcMPK6 in Arabidopsis mpk3 mpk6 mutant rescued the deficiency in floral organ abscission, while silencing of LcMPK3 or LcMPK6 in litchi significantly decreased fruitlet abscission. Importantly, a total of 49 proteins interacting with LcMPK3 were identified through yeast two-hybrid screening, including two components of the MAPK signaling cascade, five transcription factors, and two aquaporins. Furthermore, the interaction between LcMPK3/6 with LcBZR1/2, core components in brassinosteroids signaling that suppress litchi fruitlet abscission, was confirmed using in vitro and in vivo assays. Moreover, phos-tag assays demonstrated that LcMPK3/6 could phosphorylate LcBZR1/2, with several phosphorylation residues identified. Together, our findings suggest that LcMPK3 and LcMPK6 play a positive regulatory role in fruitlet abscission in litchi, and offer crucial information for the investigation of mechanisms underlying MPK3/6-mediated organ abscission in plants.

Abstract Mo086: Functional impact of phosphorylation-site mutations on PKA response of Kir2.1

Background: Kir2.1 channels maintain the cardiac resting membrane potential and assist in phase 3 repolarization. PKA-mediated phosphorylation modulates Kir2.1 function, impacting heart excitability and rhythm. Using as mass-spetometry based integrated top-down and bottom up proteomic approach, we identified five novel and confirmed a 6 th phosphorylation site. However, which of these residues impact channel function has not been previously identified. Methods: HEK293 cells were engineered to stably express either WT-Kir2.1. Ablation of the phosphorylation site was achieved with an alanine substitution of the PKA phosphorylation residue (S13A, S14A, S313A,Y326A, T347A, and S425A). Whole-cell patch clamp experiments of WT-Kir2.1 or the phosphorylation mutants were performed using a standard protocol. Following the initial recording, cells were perfused with PKA-stimulating cocktail (20µM Forskolin and 10µ IBMX) for 4 minutes and the protocol was repeated. Clampfit 10.7, GraphPad Prism, and Origin 20 were used for Data Analysis. Results: HEK293 cells stabling expressing Kir2.1 demonstrate typical inward rectification with a maximum outward current at -50mV and responds to PKA-cocktail with 2.0 pA/pF increase in maximum outward current. Both S13A and S313A had a normal response PKA stimulation. In contrast, ablation of S425 and S14 resulted in no PKA response. T347A showed little response to PKA; however, the current density is very low at baseline, indicating that this residue is necessary for normal Kir2.1 current density. The Y326 is known to be critical for normal protein folding, and ablation of the site resulted in a complete loss of function. Discussion: Of the 6 PKA sites identified by a proteomic approach, only S425 and S14 functionally influence Kir2.1 response to PKA stimulation. Further molecular dynamics simulation investigation will provide deeper insight into the conformational changes of Kir2.1 in response to phosphorylation at these sites and the relationship of channel phosphorylation with clinical channel mutation residues.

Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system.

The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.

TORC2 is required for the accumulation of γH2A in response to DNA damage

TOR protein kinases serve as the catalytic subunit of the TORC1 and TORC2 complexes, which regulate cellular growth, proliferation and survival. In the fission yeast, Schizosaccharomyces pombe, cells lacking TORC2 or its downstream kinase Gad8 (AKT or SGK1 in human cells) exhibit sensitivity to a wide range of stress conditions, including DNA damage stress. One of the first responses to DNA damage is the phosphorylation of C-terminal serine residues within histone H2AX in human cells (γH2AX), or histone H2A in yeast cells (γH2A). The kinases responsible for γH2A in S. pombe are the two DNA damage checkpoint kinases Rad3 and Tel1 (ATR and ATM, respectively, in human cells). Here we report that TORC2-Gad8 signaling is required for accumulation of γH2A in response to DNA damage and during quiescence. Using the TOR specific inhibitor, Torin1, we demonstrate that the effect of TORC2 on γH2A in response to DNA damage is immediate, rather than adaptive. The lack of γH2A is restored by deletion mutations of transcription and chromatin modification factors, including loss of components of Paf1C, SAGA, Mediator and the bromo-domain proteins Bdf1/Bdf2. Thus, we suggest that TORC2-Gad8 may affect the accumulation of γH2A by regulating chromatin structure and function.