Threonine Phosphorylation Research Articles

NEK10 kinase ablation affects mitochondrial morphology, function and protein phosphorylation status

BackgroundNEK10, a serine/threonine/tyrosine kinase belonging to the NEK (NIMA-related kinases) family, has been associated with diverse cellular processes. However, no specific target pathways have been identified. Our previous work knocking down NEK10 in HeLa cells suggested a functional association with mitochondria, as we observed altered mitochondrial morphology, mitochondrial oxygen consumption, mtDNA integrity, and reactive oxygen species levels.MethodsTo better understand this association, we studied human HAP1 cells fully knockout for NEK10 and confirmed that NEK10 has an important role in mitochondrial homeostasis.We performed the study of mitochondrial respiration, mitochondrial morphology, mitochondrial mass, and mtDNA analysis. Additionally, we showed proteome and phosphoproteome data of crude mitochondrial fraction of Parental and NEK10 KO cells using liquid chromatography-mass spectrometry (LC–MS/MS).ResultsIn the absence of NEK10 several mitochondrial functions were disturbed. Moreover, proteome and phosphoproteome analyses of mitochondrial fractions showed that NEK10 alters the threonine phosphorylation status of several mitochondrial/endoplasmic reticulum components, including HSP60, NDUFB4, and TOM20. These changes impacted the steady-state levels of a larger group of proteins, preferentially involving respiratory complexes and autophagy pathways.ConclusionWe concluded that NEK10 plays a key role in mitochondrial function, possibly by modulating the phosphorylation status of mitochondrial proteins.

Bioinformatics Analysis of the Panax ginseng Cyclophilin Gene and Its Anti-Phytophthora cactorum Activity.

In this paper, Panax ginseng cyclophilin (PgCyP) was successfully obtained through a genetic engineering technique. A bioinformatics method was used to analyze the physicochemical properties and structure of PgCyP. The results showed that PgCyP belongs to the cyclophilin gene family. The protein encoded by the PgCyP gene contains the active site of PPIase (R62, F67, and H133) and a binding site for cyclosporine A (W128). The relative molecular weight of PgCyP is 187.11 bp; its theoretical isoelectric point is 7.67, and it encodes 174 amino acids. The promoter region of PgCyP mainly contains the low-temperature environmental stress response (LTR) element, abscisic acid-responsive cis-acting element (ABRE), and light-responsive cis-acting element (G-Box). PgCyP includes a total of nine phosphorylation sites, comprising four serine phosphorylation sites, three threonine phosphorylation sites, and two tyrosine phosphorylation sites. PgCyP was recombined and expressed in vitro, and its recombinant expression was investigated. Furthermore, it was found that the recombinant PgCyP protein could effectively inhibit the germination of Phytophthora cactorum spores and the normal growth of Phytophthora cactorum mycelia in vitro. Further experiments on the roots of susceptible Arabidopsis thaliana showed that the PgCyP protein could improve the resistance of arabidopsis to Phytophthora cactorum. The findings of this study provide a basis for the use of the PgCyP protein as a new type of green biopesticide.

PKC-mediated phosphorylation governs the stability and function of CELF1 as a driver of EMT in breast epithelial cells

Epithelial to mesenchymal transition (EMT) is believed to be a principal factor contributing to cancer metastasis. The post-transcriptional and post-translational mechanisms underlying EMT are comparatively underexplored. We previously demonstrated that the CELF1 RNA binding protein is necessary and sufficient to drive the EMT of breast epithelial cells, and that the relative protein expression of CELF1 in this context was dictated at the post-translational level. Here, we elucidate the mechanism of this regulation. Mass spectrometric analysis of CELF1 isolated from mesenchymal MCF-10A cells identified multiple sites of serine and threonine phosphorylation on the protein, correlating with the increased stability of this protein in this cellular state. Analysis of phosphomimetic and serine/threonine-to-alanine phosphomutant variants of CELF1 revealed that these phosphorylation sites indeed dictate CELF1 stability, ubiquitination state, and function in vitro. Via co-immunoprecipitation and in vitro kinase assays, we identified the Protein Kinase C (PKC) alpha and epsilon isozymes as the kinases responsible for CELF1 phosphorylation in a breast cell line. Genetic epistasis experiments confirmed that these PKCs function upstream of CELF1 in this EMT program, and CELF1 phosphorylation impacts tumor metastasis in a xenograft model. This work is the first to formally establish the mechanisms underlying post-translational control of CELF1 expression and function during EMT of breast epithelial cells. Given the broad dysregulation of CELF1 expression in human breast cancer, our results may ultimately provide knowledge that may be leveraged for novel therapeutic interventions in this context.

PhosBERT: A self-supervised learning model for identifying phosphorylation sites in SARS-CoV-2-infected human cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded RNA virus, which mainly causes respiratory and enteric diseases and is responsible for the outbreak of coronavirus disease 19 (COVID-19). Numerous studies have demonstrated that SARS-CoV-2 infection will lead to a significant dysregulation of protein post-translational modification profile in human cells. The accurate recognition of phosphorylation sites in host cells will contribute to a deep understanding of the pathogenic mechanisms of SARS-CoV-2 and also help to screen drugs and compounds with antiviral potential. Therefore, there is a need to develop cost-effective and high-precision computational strategies for specifically identifying SARS-CoV-2-infected phosphorylation sites. In this work, we first implemented a custom neural network model (named PhosBERT) on the basis of a pre-trained protein language model of ProtBert, which was a self-supervised learning approach developed on the Bidirectional Encoder Representation from Transformers (BERT) architecture. PhosBERT was then trained and validated on serine (S) and threonine (T) phosphorylation dataset and tyrosine (Y) phosphorylation dataset with 5-fold cross-validation, respectively. Independent validation results showed that PhosBERT could identify S/T phosphorylation sites with high accuracy and AUC (area under the receiver operating characteristic) value of 81.9% and 0.896. The prediction accuracy and AUC value of Y phosphorylation sites reached up to 87.1% and 0.902. It indicated that the proposed model was of good prediction ability and stability and would provide a new approach for studying SARS-CoV-2 phosphorylation sites.

Acetylation of SAMHD1 at lysine 580 is crucial for blocking HIV-1 infection.

In humans, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1) is a dNTPase enzyme that prevents HIV-1 infection in non-cycling cells, such as differentiated THP-1 cells and human primary macrophages. Although phosphorylation of threonine 592 (T592) in SAMHD1 is recognized as the primary regulator of the ability to prevent HIV-1 infection, the contributions of SAMHD1 acetylation to this ability remain unknown. Mass spectrometry analysis of SAMHD1 proteins derived from cycling and non-cycling THP-1 cells, primary cycling B cells, and primary macrophages revealed that SAMHD1 is preferentially acetylated at lysine residues 354, 494, and 580 (K354, K494, and K580). In non-cycling cells, SAMHD1 is preferentially acetylated at K580, suggesting that this post-translational modification may contribute to the ability of SAMHD1 to block HIV-1 infection. Consistent with this finding, we found that mutations in K580 disrupted the ability of SAMHD1 to block HIV-1 infection without affecting the ability of SAMHD1 to deplete cellular dNTP levels. Gene editing of SAMHD1 in macrophage-like cells revealed that an intact K580 is required for HIV-1 restriction. This finding suggests that K580 acetylation in SAMHD1 is essential for blocking HIV-1 infection. More importantly, we found that a larger proportion of SAMHD1 featuring K580 acetylation could be detected in human primary macrophages when compared to human primary monocytes. In agreement, we found that SAMHD1 is acetylated during the monocyte-to-macrophage differentiation process. This finding agrees with the idea that the blockade of HIV-1 infection in macrophages requires SAMHD1 acetylation.IMPORTANCEThe natural inhibitor of HIV-1, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1), plays a pivotal role in preventing HIV-1 infection of macrophages and dendritic cells, which are vital components of the immune system. This study unveils that SAMHD1 undergoes post-translational modifications, specifically acetylation at lysines 354, 494, and 580. Our research underscores the significance of these modifications, demonstrating that acetylation at residue K580 is indispensable for SAMHD1's efficacy in blocking HIV-1 infection. Notably, K580 is found in a critical regulatory domain of SAMHD1, highlighting acetylation as a novel layer of SAMHD1 regulation for HIV-1 restriction in humans. A comprehensive understanding of the regulation mechanisms governing this anti-HIV-1 protein is crucial for leveraging nature's defense mechanisms against HIV-1 and could pave the way for innovative therapeutic strategies.

Darpp-32 is regulated by dopamine and is required for the formation of GABAergic neurons in the developing telencephalon

DARPP-32 (dopamine and cAMP-regulated phosphoprotein Mr. 32 kDa) is a phosphoprotein that is modulated by multiple receptors integrating intracellular pathways and playing roles in various physiological functions. It is regulated by dopaminergic receptors through the cAMP/protein kinase A (PKA) pathway, which modulates the phosphorylation of threonine 34 (Thr34). When phosphorylated at Thr34, DARPP-32 becomes a potent protein phosphatase-1 (PP1) inhibitor. Since dopamine is involved in the development of GABAergic neurons and DARPP-32 is expressed in the developing brain, it is possible that DARPP-32 has a role in GABAergic neuronal development. We cloned the zebrafish darpp-32 gene (ppp1r1b) gene and observed that it is evolutionarily conserved in its inhibitory domain (Thr34 and surrounding residues) and the docking motif (residues 7–11 (KKIQF)). We also characterized darpp-32 protein expression throughout the 5 days post-fertilization (dpf) zebrafish larval brain by immunofluorescence and demonstrated that darpp-32 is mainly expressed in regions that receive dopaminergic projections (pallium, subpallium, preoptic region, and hypothalamus). We demonstrated that dopamine acutely suppressed darpp-32 activity by reducing the levels of p-darpp-32 in the 5dpf zebrafish larval brain. In addition, the knockdown of darpp-32 resulted in a decrease in the number of GABAergic neurons in the subpallium of the 5dpf larval brain, with a concomitant increase in the number of DAergic neurons. Finally, we demonstrated that darpp-32 downregulation during development reduced the motor behavior of 5dpf zebrafish larvae. Thus, our observations suggest that darpp-32 is an evolutionarily conserved regulator of dopamine receptor signaling and is required for the formation of GABAergic neurons in the developing telencephalon.

CaMKIV-Mediated Phosphorylation Inactivates Freud-1/CC2D1A Repression for Calcium-Dependent 5-HT1A Receptor Gene Induction.

Calcium calmodulin-dependent protein kinase (CaMK) mediates calcium-induced neural gene activation. CaMK also inhibits the non-syndromic intellectual disability gene, Freud-1/CC2D1A, a transcriptional repressor of human serotonin-1A (5-HT1A) and dopamine-D2 receptor genes. The altered expression of these Freud-1-regulated genes is implicated in mental illnesses such as major depression and schizophrenia. We hypothesized that Freud-1 is blocked by CaMK-induced phosphorylation. The incubation of purified Freud-1 with either CaMKIIα or CaMKIV increased Freud-1 phosphorylation that was partly prevented in Freud-1-Ser644Ala and Freud-1-Thr780Ala CaMK site mutants. In human SK-N-SH neuroblastoma cells, active CaMKIV induced the serine and threonine phosphorylation of Freud-1, and specifically increased Freud-1-Thr780 phosphorylation in transfected HEK-293 cells. The activation of purified CaMKIIα or CaMKIV reduced Freud-1 binding to its DNA element on the 5-HT1A and dopamine-D2 receptor genes. In SK-N-SH cells, active CaMKIV but not CaMKIIα blocked the Freud-1 repressor activity, while Freud-1 Ser644Ala, Thr780Ala or dual mutants were resistant to inhibition by activated CaMKIV or calcium mobilization. These results indicate that the Freud-1 repressor activity is blocked by CaMKIV-induced phosphorylation at Thr780, resulting in the up-regulation of the target genes, such as the 5-HT1A receptor gene. The CaMKIV-mediated inhibition of Freud-1 provides a novel de-repression mechanism to induce 5-HT1A receptor expression for the regulation of cognitive development, behavior and antidepressant response.

Identification of a patient tissue-based phosphorylation site as a biomarker for epidermal growth factor receptor (EGFR) activity in non-small cell lung cancer.

3143 Background: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths globally. EGFR tyrosine kinase inhibitors (TKIs) have shown promise in treating NSCLC, yet resistance to these agents is common and current biomarkers rely on genetic data alone. Therefore, identifying reliable biomarkers to more comprehensively monitor EGFR activity and predict therapeutic responses is paramount. Here, we report an uncharacterized threonine phosphorylation site within core regulator of the EGFR pathway SHP2, that is strongly predictive of EGFR activity as defined by genetic mutation status that shows potential as a clinically relevant biomarker. Methods: Using DIA mass spectrometry phosphoproteomics, we could quantify >20,000 phosphosites from patient normal adjacent tumor (NAT) and primary tumor tissue. We further characterized these tissues using whole genome sequencing allowing us to define the mutational status of the EGFR. Statistical analysis was undertaken across all phospho-sites that identified a single uncharacterized phospho-site as a strong predictor of EGFR kinase activity. Finally, we generated monoclonal antibodies against the phospho-peptide and tested these in patient FFPE primary tumor tissue using immunohistochemistry to correlate phosphorylation levels with EGFR activation status. Results: Our results highlight how an individual phosphosite on SHP2 closely correlates with EGFR activity as defined by genetic mutation status in patient tissue. Interestingly, we further identify a group of patients who carry wild type EGFR yet have a phospho-signal potentially indicative of high EGFR activation without genetic aberration. These results were corroborated by a phospho-site specific antibody in FFPE tissue IHC, with strong signals observed in patients with an activating EGFR mutation (n=5), and complete absence of signal in patients harboring a wild type EGFR and no detectable mass spec signal (n=5). Conclusions: Our findings suggest that this uncharacterised SHP2 phosphosite may serve as a potent biomarker for EGFR activity in NSCLC. Further validation of the monoclonal antibodies as tools for this clinically relevant biomarker is required in larger patient cohorts to elucidate the role of the phosphosite in therapy response. This novel biomarker has the potential to guide therapeutic decision-making and contribute to personalized medicine strategies for NSCLC patients.

On the covalent nature of lysine polyphosphorylation

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville etal. (2024) Matters Arising paper, published concurrently in Molecular Cell.

Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Design, synthesis and biological evaluation of novel 1,2,4a,5-tetrahydro-4H-benzo[b][1,4]oxazino[4,3-d][1,4]oxazine-based AAK1 inhibitors with anti-viral property against SARS-CoV-2

Coronavirus entry into host cells hinges on the interaction between the spike glycoprotein of the virus and the cell-surface receptor angiotensin-converting enzyme 2 (ACE2), initiating the subsequent clathrin-mediated endocytosis (CME) pathway. AP-2-associated protein kinase 1 (AAK1) holds a pivotal role in this pathway, regulating CME by modulating the phosphorylation of the μ subunit of adaptor protein 2 (AP2M1). Herein, we report a series of novel AAK1 inhibitors based on previously reported 1,2,4a,5-tetrahydro-4H-benzo[b] [1,4]oxazino[4,3-d] [1,4]oxazine scaffold. Among 23 synthesized compounds, compound 12e is the most potent one with an IC50 value of 9.38 ± 0.34 nM against AAK1. The in vitro antiviral activity of 12e against SARS-CoV-2 was evaluated using a model involving SARS-CoV-2 pseudovirus infecting hACE2-HEK293 host cells. The results revealed that 12e was superior in vitro antiviral activity against SARS-CoV-2 entry into host cells when compared to SGC-AAK1-1 and LX9211, and its activity was comparable to that of a related and reference compound 8. Mechanistically, all AAK1 inhibitors attenuated AAK1-induced phosphorylation of AP2M1 threonine 156 and disrupted the direct interaction between AP2M1 and ACE2, ultimately inhibiting SARS-CoV-2 infection. Notably, compounds 8 and 12e exhibited a more potent effect in suppressing the phosphorylation of AP2M1 T156 and the interaction between AP2M1 and ACE2. In conclusion, novel AAK1 inhibitor 12e demonstrates significant efficacy in suppressing SARS-CoV-2 infection, and holds promise as a potential candidate for developing novel antiviral drugs against SARS-CoV-2 and other coronavirus infections.

Myelin basic proteins charge isomers interact differently with the peptidyl arginine deiminase-2.

The deamination of arginine and its conversion to citrulline is a modification observed in positively charged proteins such as histones or myelin basic protein (MBP). This reaction is catalyzed by peptidyl arginine deiminase (PAD), whose abnormal activation is associated with autoimmune diseases like rheumatoid arthritis and multiple sclerosis. However, the mechanisms that trigger PAD activation and the pathophysiological processes involved in hypercitrullination remain unknown. In this study, we investigated the interaction between PAD and various charged isomers of MBP, each differing in the degree of post-translational modification. Immunoprecipitation experiments were conducted to examine the binding between PAD and the different charge isomers of MBP. Our findings revealed that the phosphorylated forms of MBP (C3 and C4) exhibited a higher affinity for PAD compared to the unmodified (C1) and fully citrullinated forms (C8). Additionally, we observed that only in the presence of the unmodified C1 isomer did PAD undergo autocitrullination, which was inhibited by the endogenous guanidine-containing component, creatine. In the presence of other isomers, PAD did not undergo autocitrullination. Furthermore, we found that the unmodified isomer of MBP-C1 contains methylated arginines, which were not affected by the pre-treatment with PAD. Based on our findings, we propose that the increased phosphorylation of central threonines in the original MBP may trigger PAD activation, leading to increased citrullination of the protein and subsequent disorganization of the myelin sheath. These insights contribute to a better understanding of the underlying mechanisms in autoimmune diseases associated with hypercitrullination, potentially opening new avenues for therapeutic interventions.

In-silico investigation of RPS6KB1 associated cancer inhibitor: a drug repurposing study

The ribosomal protein S6 kinase beta-1 (RPS6KB1), also known as p70S6 kinase, plays a crucial role in various disease-related conditions such as diabetes, obesity, and cancer. Its activity is regulated by phosphorylation events, including phosphorylation of Threonine 389 in the hydrophobic motif by the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of Threonine 229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1). However, other phenomena connected to RPS6KB1 remain unknown. In this study, we employed virtual screening and molecular docking techniques on the molecules in the ZINC library to identify potential inhibitors. Molecular dynamics (MD) simulations and MMGBSA calculations were carried out on promising compounds to determine their binding affinity and stability. We also evaluated the drug-likeness properties of the selected compounds. A comparative study between the native RPS6KB1 structure, co-crystal ligands, and the shortlisted molecules from the ZINC dataset was carried out. The identified molecules possess significant potential for future in vitro and in vivo studies, paving the way for developing effective cancer treatments. Communicated by Ramaswamy H. Sarma

Drosophila Smad2 degradation occurs independently of linker phosphorylations.

TGF-β signals are important for proliferation, differentiation, and cell fate determination during embryonic development and tissue homeostasis in adults. Drosophila Activin/TGF-β signals are transduced intracellularly when its transcription factor dSmad2 (also called Smad on X or Smox) is C-terminally phosphorylated by pathway receptors. Recently, it has been shown that receptor-activated dSmad2 undergoes bulk degradation, however, the mechanism of how this occurs is unknown. Here we investigated if two putative linker phosphorylation sites are involved in dSmad2 degradation. We demonstrate that degradation of activated-dSmad2 occurs independently of threonine phosphorylation at linker sites 252 and 277. We also show that dSmad2 degradation is not carried out by cellular proteasomes.

Post-translational modulation of cell signalling through protein succinylation

Cells need to adapt their activities to extra- and intracellular signalling cues. To translate a received extracellular signal, cells have specific receptors that transmit the signal to downstream proteins so that it can reach the nucleus to initiate or repress gene transcription. Post-translational modifications (PTMs) of proteins are reversible or irreversible chemical modifications that help to further modulate protein activity. The most commonly observed PTMs are the phosphorylation of serine, threonine, and tyrosine residues, followed by acetylation, glycosylation, and amidation. In addition to PTMs that involve the modification of a certain amino acid (phosphorylation, hydrophobic groups for membrane localisation, or chemical groups like acylation), or the conjugation of peptides (SUMOylation, NEDDylation), structural changes such as the formation of disulphide bridge, protein cleavage or splicing can also be classified as PTMs. Recently, it was discovered that metabolites from the tricarboxylic acid (TCA) cycle are not only intermediates that support cellular metabolism but can also modify lysine residues. This has been shown for acetate, succinate, and lactate, among others. Due to the importance of mitochondria for the overall fitness of organisms, the regulatory function of such PTMs is critical for protection from aging, neurodegeneration, or cardiovascular disease. Cancer cells and activated immune cells display a phenotype of accelerated metabolic activity known as the Warburg effect. This metabolic state is characterised by enhanced glycolysis, the use of the pentose phosphate pathway as well as a disruption of the TCA cycle, ultimately causing the accumulation of metabolites like citrate, succinate, and malate. Succinate can then serve as a signalling molecule by directly interacting with proteins, by binding to its G protein-coupled receptor 91 (GPR91) and by post-translationally modifying proteins through succinylation of lysine residues, respectively. This review is focus on the process of protein succinylation and its importance in health and disease.

A papain-like cysteine protease-released small signal peptide confers wheat resistance to wheat yellow mosaic virus

Wheat yellow mosaic virus (WYMV), a soil-borne pathogen, poses a serious threat to global wheat production. Here, we identify a WYMV resistance gene, TaRD21A, that belongs to the papain-like cysteine protease family. Through genetic manipulation of TaRD21A expression, we establish its positive role in the regulation of wheat to WYMV resistance. Furthermore, our investigation shows that the TaRD21A-mediated plant antiviral response relies on the release of a small peptide catalyzed by TaRD21A protease activity. To counteract wheat resistance, WYMV-encoded nuclear inclusion protease-a (NIa) suppress TaRD21A activity to promote virus infection. In resistant cultivars, a natural variant of TaRD21A features a alanine to serine substitution and this substitution enables the phosphorylation of Serine, thereby weakening the interaction between NIa and TaRD21A, reinforcing wheat resistance against WYMV. Our study not only unveils a WYMV resistance gene but also offers insights into the intricate mechanisms underpinning resistance against WYMV.

Upregulation of LRRK2 following traumatic brain injury does not directly phosphorylate Thr175 tau.

Phosphorylated microtubule-associated protein tau (tau) aggregates are a pathological hallmark of various neurodegenerative diseases, including chronic traumatic encephalopathy and amyotrophic lateral sclerosis with cognitive impairment. While there are many residues phosphorylated on tau, phosphorylation of threonine 175 (pThr175 tau) has been shown to initiate fibril formation in vitro and is present in pathological tau aggregates in vivo. Given this, preventing Thr175 tau phosphorylation presents a potential approach to reduce fibril formation; however, the kinase(s) acting on Thr175 are not yet fully defined. Using a single controlled cortical impact rodent model of traumatic brain injury (TBI), which rapidly induces Thr175 tau phosphorylation, we observed an upregulation and alteration in subcellular localization of leucine-rich repeat kinase 2 (LRRK2), a kinase that has been implicated in tau phosphorylation. LRRK2 upregulation was evident by one-day post-injury and persisted to day 10. The most notable changes were observed in microglia at the site of injury in the cortex. To determine if the appearance of pThr175 tau was causally related to the upregulation of LRRK2 expression, we examined the ability of LRRK2 to phosphorylate Thr175in vitro by co-transfecting 2N4R human WT-tau with either LRRK2-WT, constitutively-active LRRK2-G2019S or inactive LRRK2-3XKD. We found no significant difference in the level of pThr175 tau between the overexpression of LRRK2-WT, -G2019S or -3XKD, suggesting LRRK2 does not phosphorylate tau at Thr175. Further, downstream events known to follow Thr175 phosphorylation and known to be associated with pathological tau fibril formation (pSer9-GSK3β and pThr231 tau induction) also remained unchanged. We conclude that while LRRK2 expression is altered in TBI, it does not contribute directly to pThr175 tau generation.

Assessing the Role of Post-Translational Modifications of Mitochondrial RNA Polymerase.

The mitochondrial proteome is subject to abundant post-translational modifications, including lysine acetylation and phosphorylation of serine, threonine, and tyrosine. The biological function of the majority of these protein modifications is unknown. Proteins required for the transcription and translation of mitochondrial DNA (mtDNA) are subject to modification. This suggests that reversible post-translational modifications may serve as a regulatory mechanism for mitochondrial gene transcription, akin to mechanisms controlling nuclear gene expression. We set out to determine whether acetylation or phosphorylation controls the function of mitochondrial RNA polymerase (POLRMT). Mass spectrometry was used to identify post-translational modifications on POLRMT. We analyzed three POLRMT modification sites (lysine 402, threonine 315, threonine 993) found in distinct structural regions. Amino acid point mutants that mimic the modified and unmodified forms of POLRMT were employed to measure the effect of acetylation or phosphorylation on the promoter binding ability of POLRMT in vitro. We found a slight decrease in binding affinity for the phosphomimic at threonine 315. We did not identify large changes in viability, mtDNA content, or mitochondrial transcript level upon overexpression of POLRMT modification mimics in HeLa cells. Our results suggest minimal biological impact of the POLRMT post-translational modifications studied in our system.

Analysis of Structural Changes in the Protein near the Phosphorylation Site.

Modification of the protein after synthesis (PTM) often affects protein function as supported by numerous studies. However, there is no consensus about the degree of structural protein changes after modification. For phosphorylation of serine, threonine, and tyrosine, which is a common PTM in the biology of living organisms, we consider topical issues related to changes in the geometric parameters of a protein (Rg, RMSD, Cα displacement, SASA). The effect of phosphorylation on protein geometry was studied both for the whole protein and at the local level (i.e., in different neighborhoods of the modification site). Heterogeneity in the degree of protein structural changes after phosphorylation was revealed, which allowed for us to isolate a group of proteins having pronounced local structural changes in the neighborhoods of up to 15 amino acid residues from the modification site. This is a comparative study of protein structural changes in neighborhoods of 3-15 amino acid residues from the modified site. Amino acid phosphorylation in proteins with pronounced local changes caused switching from the inactive functional state to the active one.

The Disruption of a Nuclear Export Signal in the C-Terminus of the Herpes Simplex Virus 1 Determinant of Pathogenicity UL24 Protein Leads to a Syncytial Plaque Phenotype.

UL24 of herpes simplex virus 1 (HSV-1) has been shown to be a determinant of pathogenesis in mouse models of infection. The N-terminus of UL24 localizes to the nucleus and drives the redistribution of nucleolin and B23. In contrast, when expressed alone, the C-terminal domain of UL24 accumulates in the Golgi apparatus; its importance during infection is unknown. We generated a series of mammalian expression vectors encoding UL24 with nested deletions in the C-terminal domain. Interestingly, enhanced nuclear staining was observed for several UL24-deleted forms in transient transfection assays. The substitution of a threonine phosphorylation site had no effect on UL24 localization or viral titers in cell culture. In contrast, mutations targeting a predicted nuclear export signal (NES) significantly enhanced nuclear localization, indicating that UL24 is able to shuttle between the nucleus and the cytoplasm. Recombinant viruses that encode UL24-harboring substitutions in the NES led to the accumulation of UL24 in the nucleus. Treatment with the CRM-1-specific inhibitor leptomycin B blocked the nuclear export of UL24 in transfected cells but not in the context of infection. Viruses encoding UL24 with NES mutations resulted in a syncytial phenotype, but viral yield was unaffected. These results are consistent with a role for HSV-1 UL24 in late cytoplasmic events in HSV-1 replication.