Protein Post-translational Modifications Research Articles

Quantitative analysis of non-histone lysine methylation sites and lysine demethylases in breast cancer cell lines.

Growing evidence shows that lysine methylation is a widespread protein post-translational modification that regulates protein function on histone and non-histone proteins. Numerous studies have demonstrated that dysregulation of lysine methylation mediators contributes to cancer growth and chemotherapeutic resistance. While changes in histone methylation are well documented with extensive analytical techniques available, there is a lack of high-throughput methods to reproducibly quantify changes in the abundances of the mediators of lysine methylation and non-histone lysine methylation (Kme) simultaneously across multiple samples. Recent studies by our group and others have demonstrated that antibody enrichment is not required to detect lysine methylation, prompting us to investigate the use of Tandem Mass Tag (TMT) labeling for global Kme quantification sans antibody enrichment in four different breast cancer cell lines (MCF-7, MDA-MB-231, HCC1806, and MCF10A). To improve the quantification of KDMs, we incorporated a lysine demethylase (KDM) isobaric trigger channel, which enabled 96% of all KDMs to be quantified while simultaneously quantifying 326 Kme sites. Overall, 142 differentially abundant Kme sites and eight differentially abundant KDMs were identified between the four cell lines, revealing cell line-specific patterning.

O-GlcNAc modification of HSP27 alters its protein interactions and promotes refolding of proteins through the BAG3/HSP70 co-chaperone.

Almost all types of cellular stress induce post-translational O-GlcNAc modifications of proteins, and this increase promotes cell survival. We previously demonstrated that O-GlcNAc on certain small heat shock proteins (sHSPs), including HSP27, directly increases their chaperone activity as one potential protective mechanism. Here, we furthered our use of synthetic proteins to prepare biotinylated sHSPs and show that O-GlcNAc modification of HSP27 also changes how it interacts within the sHSP system and the broader HSP network. Specifically, we show that O-GlcNAc modified HSP27 binds more strongly to the co-chaperone protein BAG3, which then promotes refolding of a model substrate by HSP70. We use proteomics to identify other potential HSP27 interactions that are changed by O-GlcNAc, including one that we confirm with another sHSP, αB-crystallin. These findings add additional evidence for O-GlcNAc as a switch for regulating protein-protein interactions and for modifications of chaperones as one mechanism by which O-GlcNAc protects against protein aggregation.

Disruption of herpes simplex virus type 2 pUL21 phosphorylation impairs secondary envelopment of cytoplasmic nucleocapsids.

It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species.

Decoding polyubiquitin regulation of KV7. 1 functional expression with engineered linkage-selective deubiquitinases.

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the 'ubiquitin code' that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.

Activation of D2-like dopamine receptors improves the neuronal network and cognitive function of PPT1KI mice.

Palmitoyl-protein thioesterase 1 (PPT1) is a lysosomal depalmitoylation enzyme that mediates protein posttranslational modifications. Loss-of-function mutation of PPT1 causes a failure of the lysosomal degradation of palmitoylated proteins and results in a congenital disease characterized by progressive neuronal degeneration referred to as infantile neuronal ceroid lipofuscinosis (INCL). A mouse knock-in model of PPT1 (PPT1-KI) was established by introducing the R151X mutation into exon 5 of the PPT1 gene, which exhibited INCL-like pathological lesions. We previously reported that hippocampal γ oscillations were impaired in PPT1 mice. Hippocampal γ oscillations can be enhanced by selective activation of the dopamine D4 receptor (DR4), a dopamine D2-like receptor. In this study, we investigated the changes in DR expression and the effects of dopamine and various DR agonists on neural network activity, cognition and motor function in PPT1KI mice. Cognition and motor defects were evaluated via Y-maze, novel object recognition and rotarod tests. Extracellular field potentials were elicited in hippocampal slices, and neuronal network oscillations in the gamma frequency band (γ oscillations) were induced by perfusion with kainic acid (200 nM). PPT1KI mice displayed progressive impairments in γ oscillations and hippocampus-related memory, as well as abnormal expression profiles of dopamine receptors with preserved expression of DR1 and 3, increased membrane expression of DR4 and decreased DR2 levels. The immunocytochemistry analysis revealed the colocalization of PPT1 with DR4 or DR2 in the soma and large dendrites of both WT and PPT1KI mice. Immunoprecipitation confirmed the interaction between PPT1 and DR4 or DR2. The impaired γ oscillations and cognitive functions were largely restored by the application of exogenous dopamine, the selective DR2 agonist quinpirole or the DR4 agonist A412997. Furthermore, the administration of A412997 (0.5 mg/kg, i.p.) significantly upregulated the activity of CaMKII in the hippocampus of 5-month-old PPT1KI mice. Collectively, these results suggest that the activation of D2-like dopamine receptors improves cognition and network activity in PPT1KI mice and that specific DR subunits may be potential targets for the intervention of neurodegenerative disorders, such as INCL.

Exploring Protein Post-Translational Modifications of Breast Cancer Cells Using a Novel Combinatorial Search Algorithm.

Post-translational modification of proteins plays an important role in cancer cell biology. Proteins encoded by oncogenes may be activated by phosphorylation, products of tumour suppressors might be inactivated by phosphorylation or ubiquitinylation, which marks them for degradation; chromatin-binding proteins are often methylated and/or acetylated. These are just a few of the many hundreds of post-translational modifications discovered by years of painstaking experimentation and the chemical analysis of purified proteins. In recent years, mass spectrometry-based proteomics emerged as the principal technique for identifying such modifications in samples from cultured cells and tumour tissue. Here, we used a recently developed combinatorial search algorithm implemented in the MGVB toolset to identify novel modifications in samples from breast cancer cell lines. Our results provide a rich resource of coupled protein abundance and post-translational modification data seen in the context of an important biological function-the response of cells to interferon gamma treatment-which can serve as a starting point for future investigations to validate promising modifications and explore the utility of the underlying molecular mechanisms as potential diagnostic or therapeutic targets.

Effect of β-1,4-GalTI on the biological function of astrocytes treated by LPS.

Inflammation of the central nervous system (CNS) is a common feature of neurological disorders and infections, playing a crucial role in the development of CNS-related conditions. CNS inflammation is primarily regulated by glial cells, with astrocytes being the most abundant type in the mammalian CNS. Numerous studies have demonstrated that astrocytes, as immunocompetent cells, perform diverse and complex functions in both health and disease. Glycosylation, a critical post-translational modification of proteins, regulates numerous biological functions. The expression and activity of glycosyltransferases, the enzymes responsible for glycosylation, are closely associated with the pathogenesis of various diseases. β-1,4-GalTI, a mammalian glycosyltransferase, plays a significant role in cell-cell interactions, adhesion, and migration. Although many studies have focused on β-1,4-GalTI, few have explored its effects on astrocyte function. In this study, we constructed lentiviral vectors for both interference and overexpression of β-1,4-GalTI and discovered that β-1,4-GalTI knockdown inhibited astrocyte migration and proliferation, while its overexpression promoted these processes. Concurrently, β-1,4-GalTI knockdown reduced the expression of TNF-α, IL-1β, and IL-6, whereas overexpression enhanced the expression of these cytokines. These findings suggest that modulating β-1,4-GalTI activity can influence the molecular functions of astrocytes and provide a theoretical foundation for further research into β-1,4-GalTI as a potential therapeutic target in astrocyte-mediated inflammation.

A Comprehensive Pan-Cancer Analysis Identified that TRIB3 was Associated with Immune Cell Infiltration and Poor Prognosis.

Previous studies have demonstrated that TRIB3 plays a carcinogenic role in tumor progression. However, the exploration of TRIB3 at the pan-cancer level has not been reported. This study aimed to conduct a comprehensive pan-cancer analysis of TRIB3. We explored the expression pattern and functional mechanism of TRIB3 on the basis of multiple databases. We first explored the expression level of TRIB3 in the TCGA database. Then, the receiver operation characteristic curve (ROC), Kaplan-Meier plotter, and Cox regression were used to estimate the diagnostic and prognostic value of TRIB3, respectively. We also explored the relationship between TRIB3 and the infiltration of tumor immune cells, as well as the expression of immune checkpoint molecules. Gene enrichment and protein interaction network analysis were carried out to identify possible carcinogenic molecular mechanisms and functional pathways. Finally, we compared the non-promoter region methylation of TRIB3 in normal and tumor tissues and explored potential systems with unique functions in TRIB3-mediated tumorigenesis. The expression level of TRIB3 was elevated in multiple tumor types, and the high expression of TRIB3 was associated with poor prognosis. TRIB3 had a higher frequency of genetic changes in several tumors and showed varying trends in TRIB3 methylation levels. Additionally, high expression of TRIB3 was also associated with infiltration of cancer-related fibroblasts and different types of immune cells and was positively correlated with the expression of immune checkpoint molecules. Furthermore, gene enrichment analysis suggested that TRIB3 may play a role in the malignant progression of cancer by participating in protein post-translational modifications and activating transcription initiation factors. Our pan-cancer analysis provided the potential carcinogenic role of TRIB3 in tumors and verified a promising target for clinical immune treatment.

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

Comparative Proteomic Analysis Reveals the Effect Mechanisms of Glucose on the Biomass and Phenolic Glycoside Esters Synthesis Activity of Candida Parapsilosis ACCC 20221 Whole-Cell Catalyst.

A new Candida parapsilosis ACCC 20221 (C. parapsilosis ACCC 20221) whole-cell catalyst with a high phenolic glycoside esters synthesis activity and large biomass was obtained after culture with glucose. The possible mechanisms were revealed by using comparative proteomics. It found the expression of proteins involved in post-translational modification, protein turnover, and chaperone, and RNA processing and modification was upregulated, which ensured the metabolic balance and accurate translation, correct folding, and post-translational modification of proteins, thus enhancing the production of lipases in C. parapsilosis ACCC 20221 cultured with glucose. Moreover, the glycolysis pathway, tricarboxylic acid cycle, and fatty acids synthesis were enhanced, while the β-oxidation of fatty acids was weakened in C. parapsilosis ACCC 20221 cells cultured with glucose, which led to an increase in energy generation and cell membrane synthesis; thus, large biomass was obtained. In addition, CCE40476.1 and CAC86400.1, which were likely to exert arbutin esters synthesis activity in C. parapsilosis ACCC 20221, were screened, and it was found that vinyl propionate could easily enter the catalytic pocket of CCE40476.1 and form hydrogen bonding interactions with Leu191 and Ser266.

Exploring protein N-glycosylation in the green microalga Dunaliella salina

N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum where an oligosaccharide precursor is added to the newly synthesized protein and continues in the Golgi apparatus where the N-linked carbohydrate sequences are processed. Importantly, the most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly currently produced in mammalian cells which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered as efficient and eco-friendly alternative platforms for the production of biologics. However, unlike for C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man5GlcNAc2 oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii that gives rise to the synthesis of a non-canonical oligomannoside Man5GlcNAc2. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man5GlcNAc2.

Molecular age prediction using skull bone samples from individuals with and without signs of decomposition: a multivariate approach combining analysis of posttranslational protein modifications and DNA methylation.

The prediction of the chronological age of a deceased individual at time of death can provide important information in case of unidentified bodies. The methodological possibilities in these cases depend on the availability of tissues, whereby bones are preserved for a long time due to their mineralization under normal environmental conditions. Age-dependent changes in DNA methylation (DNAm) as well as the accumulation of pentosidine (Pen) and D-aspartic acid (D-Asp) could be useful molecular markers for age prediction. A combination of such molecular clocks into one age prediction model seems favorable to minimize inter- and intra-individual variation. We therefore developed (I) age prediction models based on the three molecular clocks, (II) examined the improvement of age prediction by combination, and (III) investigated if samples with signs of decomposition can also be examined using these three molecular clocks. Skull bone from deceased individuals was collected to obtain a training dataset (n = 86), and two independent test sets (without signs of decomposition: n = 44, with signs of decomposition: n = 48). DNAm of 6 CpG sites in ELOVL2, KLF14, PDE4C, RPA2, TRIM59 and ZYG11A was analyzed using massive parallel sequencing (MPS). The D-Asp and Pen contents were analyzed by high performance liquid chromatography (HPLC). Age prediction models based on ridge regression were developed resulting in mean absolute errors (MAEs)/root mean square errors (RMSE) of 5.5years /6.6 years (DNAm), 7.7 years /9.3 years (Pen) and 11.7 years /14.6 years (D-Asp) in the test set. Unsurprisingly, a general lower accuracy for the DNAm, D-Asp, and Pen models was observed in samples from decomposed bodies (MAE: 7.4-11.8 years, RMSE: 10.4-15.4 years). This reduced accuracy could be caused by multiple factors with different impact on each molecular clock. To acknowledge general changes due to decomposition, a pilot model for a possible age prediction based on the decomposed samples as training set improved the accuracy evaluated by leave-one-out-cross validation (MAE: 6.6-12 years, RMSE: 8.1-15.9 years). The combination of all three molecular age clocks did reveal comparable MAE and RMSE results to the pure analysis of the DNA methylation for the test set without signs of decomposition. However, an improvement by the combination of all three clocks was possible for the decomposed samples, reducing especially the deviation in case of outliers in samples with very high decomposition and low DNA content. The results demonstrate the general potential in a combined analysis of different molecular clocks in specific cases.

O-GlcNAcylation mediates Wnt-stimulated bone formation by rewiring aerobic glycolysis

Wnt signaling is an important target for anabolic therapies in osteoporosis. A sclerostin-neutralizing antibody (Scl-Ab), that blocks the Wnt signaling inhibitor (sclerostin), has been shown to promote bone mass in animal models and clinical studies. However, the cellular mechanisms by which Wnt signaling promotes osteogenesis remain to be further investigated. O-GlcNAcylation, a dynamic post-translational modification of proteins, controls multiple critical biological processes including transcription, translation, and cell fate determination. Here, we report that Wnt3a either induces O-GlcNAcylation rapidly via the Ca2+-PKA-Gfat1 axis, or increases it in a Wnt-β-catenin-dependent manner following prolonged stimulation. Importantly, we find O-GlcNAcylation indispensable for osteoblastogenesis both in vivo and in vitro. Genetic ablation of O-GlcNAcylation in the osteoblast-lineage diminishes bone formation and delays bone fracture healing in response to Wnt stimulation in vivo. Mechanistically, Wnt3a induces O-GlcNAcylation at Serine 174 of PDK1 to stabilize the protein, resulting in increased glycolysis and osteogenesis. These findings highlight O-GlcNAcylation as an important mechanism regulating Wnt-induced glucose metabolism and bone anabolism.

USP33 facilitates the ovarian cancer progression via deubiquitinating and stabilizing CBX2.

Post-translational modifications of proteins play a pivotal role in both the initiation and progression of ovarian cancer. Despite the recognition of USP33 as a significant factor in various cancers, its specific function and underlying mechanisms in ovarian cancer remain elusive. Proteomics and ubiquitinomics approaches were coupled to screen novel substrate proteins directly regulated by USP33. Our findings unveil that USP33 was observed to eliminate K27- and K48-linked ubiquitin chains from CBX2 at the K277 position. Notably, acetylation of CBX2 at K199, catalyzed by lysine acetyltransferase GCN5, was found to enhance its interaction with USP33, subsequently promoting further deubiquitination and stabilization. Functionally, our experiments demonstrate that USP33 significantly enhances ovarian cancer proliferation and metastasis in a CBX2-dependent manner. Furthermore, analysis revealed a direct positive correlation between the expression levels of USP33 and CBX2 proteins in human specimens, with elevated levels being associated with reduced survival rates in ovarian cancer patients. These findings elucidate the mechanism by which USP33 augments ovarian cancer progression through the stabilization of CBX2, underscoring the USP33-CBX2 axis as a promising therapeutic target in ovarian cancer management.

The Effect of the Lysine Acetylation Modification of ClpP on the Virulence of Vibrio alginolyticus.

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus.

Species-specific model based on sequence and structural information for ubiquitination sites prediction

Ubiquitination is a common post-translational modification of proteins in eukaryotic cells, and it is also a significant method of regulating protein biological function. Computational methods for predicting ubiquitination sites can serve as a cost-effective and time-saving alternative to experimental methods. Existing computational methods often build classifiers based on protein sequence information, physical and chemical properties of amino acids, evolutionary information, and structural parameters. However, structural information about most proteins cannot be found in existing databases directly. The features of proteins differ among species, and some species have small amounts of ubiquitinated proteins. Therefore, it is necessary to develop species-specific models that can be applied to datasets with small sample sizes. To solve these problems, we propose a species-specific model (SSUbi) based on a capsule network, which integrates proteins’ sequence and structural information. In this model, the feature extraction module is composed of two sub-modules that extract multi-dimensional features from sequence and structural information respectively. In the submodule, the convolution operation is used to extract encoding dimension features, and the channel attention mechanism is used to extract feature map dimension features. After integrating the multi-dimensional features from both types of information, the species-specific capsule network further converts the features into capsule vectors and classifies species-specific ubiquitination sites. The experimental results show that SSUbi can effectively improve the prediction performance of species with small sample sizes and outperform other models.

African swine fever virus modulates the endoplasmic reticulum stress-ATF6-calcium axis to facilitate viral replication

ABSTRACT African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

Application and research progress of MultiBac: A review.

Although the traditional Escherichia coli expression system has matured and is cost-effective, the posttranslation modifications of proteins expressed in eukaryotic cells differ significantly from those expressed in E coli. Insect cells have gradually entered the realm of researchers; however, the proteins synthesized by insect cells are somewhat different from those of mammals in terms of modification. Herein, we have introduced a relatively new method. MultiBac, We introduce the development process, characteristics, and applications of MultiBac technology. And provide new methods for basic researchers. MultiBac has evolved into an indispensable tool in the fields of biotechnology and pharmaceuticals, facilitating the efficient production of recombinant proteins and the study of complex protein complexes. Furthermore, its development has benefited from the integration of synthetic biology techniques, providing additional versatility. But it also has some disadvantages. MultiBac technology is poised to become a key tool in unlocking the mysteries of the protein world, propelling the life sciences ever forward. But researchers should consider its limitations when selecting the most appropriate expression system for their specific needs.

RBN-2397, a PARP7 Inhibitor, Synergizes with Paclitaxel to Inhibit Proliferation and Migration of Ovarian Cancer Cells.

Mono(ADP-ribosyl)ation (MARylation), a post translational modification of proteins, is emerging as an important regulator of the biology of cancer cells. PARP7 (TiPARP), a mono (ADP-ribosyl) transferase (MART), MARylates its substrate α-tubulin in ovarian cancer cells, promoting destabilization of microtubules, cell growth, and migration. Recent development of RBN-2397, a potent inhibitor that selectively acts on PARP7, has provided a new tool for exploring the role of PARP7 catalytic activity in biological processes. In this study, we investigated the role of PARP7 catalytic activity in the regulation of ovarian cancer cell biology via MARylation of α-tubulin. Ovarian cancer cell lines (OVCAR4, OVCAR3) were treated with RBN-2397 and paclitaxel, both separately and in combination. Western blotting and immunoprecipitation confirmed the effects of RBN-2397 on α-tubulin MARylation and stabilization. Cell proliferation and migration were assessed, and α-tubulin stabilization was quantified using immunofluorescent imaging. RNA-sequencing was performed to assess the effects on gene expression changes. RBN-2397 inhibited PARP7 activity, decreasing α-tubulin MARylation, leading to its stabilization, and reducing cancer cell proliferation and migration. The addition of paclitaxel further enhanced these effects, highlighting a synergistic interaction between the two drugs. Mutating the site of PARP7-mediated MARylation on α-tubulin similarly resulted in microtubule stabilization and decreased cell migration in the presence of paclitaxel. This study demonstrates that targeting PARP7 with RBN-2397, particularly in combination with paclitaxel, offers an effective strategy for inhibiting aggressive ovarian cancer cell phenotypes. Our findings underscore the potential of combining PARP7 inhibitors with established chemotherapeutics to enhance treatment efficacy in ovarian cancer.

A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells.

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated on three acidic residues by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls stress granule assembly and disassembly in ovarian cancer cells.