Alterations In Post-translational Modifications Research Articles

Post-translational modifications in prion diseases.

More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone amino-acids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases.

Chemical Proteomics–Guided Discovery of Covalent Ligands for Cancer Proteins

Advances in genome sequencing and editing technologies have enriched our understanding of the biochemical pathways that drive tumorigenesis. Translating this knowledge into new medicines for cancer treatment, however, remains challenging, and many oncogenic proteins have proven recalcitrant to conventional approaches for chemical probe and drug discovery. Here, we discuss how innovations in chemical proteomics and covalent chemistry are being integrated to identify and advance first-in-class small molecules that target cancer-relevant proteins. Mechanistic studies have revealed that covalent compounds perturb protein functions in cancer cells in diverse ways that include the remodeling of protein–protein and protein–RNA complexes, as well as through alterations in posttranslational modification. We speculate on the attributes of chemical proteomics and covalent chemistry that have enabled targeting of previously inaccessible cancer-relevant pathways and consider technical challenges that remain to be addressed in order to fully realize the druggability of the cancer proteome.

Immunometabolic Maladaptations to the Tumor Microenvironment.

Tumors consist of cancer cells and a wide range of tissue resident and infiltrating cell types. Tumor metabolism, however, has largely been studied on whole tumors or cancer cells and the metabolism of infiltrating immune cells remains poorly understood. It is now clear from a range of analyses and metabolite rescue studies that metabolic adaptations to the tumor microenvironment (TME) directly impede T-cell and macrophage effector functions. The drivers of metabolic adaptation to the TME and metabolic immune suppression include depletion of essential nutrients, accumulation of waste products or immune suppression metabolites, and metabolic signaling through altered posttranslational modifications. Each infiltrating immune cell subset differs, however, with specific metabolic requirements and adaptations that can be maladaptive for antitumor immunity. Here, we review T-cell and macrophage adaptation and metabolic immune suppression in solid tumors. Ultimately, understanding and addressing these challenges will improve cancer immunotherapy and adoptive chimeric antigen receptor T-cell therapies.

Mining proteomics data to extract post-translational modifications associated with gastric cancer.

Gastric cancers are highly heterogeneous, deep-seated tumours associated with late diagnosis and poor prognosis. Post-translational modifications (PTMs) of proteins are known to be well-associated with oncogenesis and metastasis in most cancers. Several enzymes which drive PTMs have also been used as theranostics in cancers of the breast, ovary, prostate and bladder. However, there is limited data on PTMs in gastric cancers. Considering that experimental protocols for simultaneous analysis of multiple PTMs are being explored, a data-driven approach involving reanalysis of mass spectrometry-derived data is useful in cataloguing altered PTMs. We subjected publicly available mass spectrometry data on gastric cancer to an iterative searching strategy for fetching PTMs including phosphorylation, acetylation, citrullination, methylation and crotonylation. These PTMs were catalogued and further analyzed for their functional enrichment through motif analysis. This value-added approach delivered identification of 21,710 unique modification sites on 16,364 modified peptides. Interestingly, we observed 278 peptides corresponding to 184 proteins to be differentially abundant. Using bioinformatics approaches, we observed that majority of these altered PTMs/proteins belonged to cytoskeletal and extracellular matrix proteins, which are known to be perturbed in gastric cancer. The dataset derived by this mutiPTM investigation can provide leads to further investigate the potential role of altered PTMs in gastric cancer management.

Defining the Sarcomeric Proteoform Landscape in Ischemic Cardiomyopathy by Top-Down Proteomics.

Ischemic cardiomyopathy (ICM) is a prominent form of heart failure, but the molecular mechanisms underlying ICM remain relatively understudied due to marked phenotypic heterogeneity. Alterations in post-translational modifications (PTMs) and isoform switches in sarcomeric proteins play important roles in cardiac pathophysiology. Thus, it is essential to define sarcomeric proteoform landscape to better understand ICM. Herein, we have implemented a top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics method for the identification and quantification of sarcomeric proteoforms in the myocardia of donors without heart diseases (n = 16) compared to end-stage ICM patients (n = 16). Importantly, quantification of post-translational modifications (PTMs) and expression reveal significant changes in various sarcomeric proteins extracted from ICM tissues. Changes include altered phosphorylation and expression of cardiac troponin I (cTnI) and enigma homologue 2 (ENH2) as well as an increase in muscle LIM protein (MLP) and calsarcin-1 (Cal-1) phosphorylation in ICM hearts. Our results imply that the contractile apparatus of the sarcomere is severely dysregulated during ICM. Thus, this is the first study to uncover significant molecular changes to multiple sarcomeric proteins in the LV myocardia of the end-stage ICM patients using liquid chromatography-mass spectrometry (LC-MS)-based top-down proteomics. Raw data are available via the PRIDE repository with identifier PXD038066.

Altered Posttranslational Modification of Microtubules Contributes to Disturbed Enterocyte Morphology in Celiac Disease.

Celiac disease (CD) represents a frequent autoimmune disease triggered by the ingestion of gliadin in genetically predisposed individuals. The alteration of enterocytes and brush border membrane morphology have been repetitively demonstrated, but the underlying mechanisms remain unclear. Microtubules represent a major element of the cytoskeleton and exert multiple functions depending on their tyrosination status. The aim of our study was to investigate whether posttranslational modification of microtubules was altered in the context of CD and whether this mechanism contributed to morphological changes of CD enterocytes. We examined the expression of tubulin tyrosine ligase (TTL) and vasohibin-2 (VASH2) and the level of detyrosinated and acetylated tubulin in duodenal biopsies and Caco-2 cells by immunoblot and immunofluorescence microcopy. Electron microscopy was performed to investigate the subcellular distribution of detyrosinated tubulin and brush border membrane architecture in CD biopsies and Madin-Darby Canine Kidney type II (MDCK) cells lacking TTL. CD enterocytes and Caco-2 cells stimulated with digested gliadin or IFN-y displayed a flattened cell morphology. This disturbed cellular architecture was accompanied by an increased amount of detyrosinated and acetylated tubulin and corresponding high expression of VASH2 and low expression of TTL. The altered posttranslational modification of tubulin was reversible after the introduction of the gluten-free diet. CD enterocytes and MDCK cells deficient in TTL displayed a reduced cell height along with an increased cell width and a reduced number of apical microvilli. Our results provide a functional explanation for the observed morphological alterations of the enterocytes observed in CD and provide diagnostic potential of the tyrosination status of microtubules as an early marker of villous atrophy and CD inflammation.

Dynamic regulation of eEF1A1 acetylation affects colorectal carcinogenesis.

The dysregulation of the translation elongation factor families which are responsible for reprogramming of mRNA translation has been shown to contribute to tumor progression. Here, we report that the acetylation of eukaryotic Elongation Factor 1 Alpha 1 (eEF1A1/EF1A1) is required for genotoxic stress response and maintaining themalignancy of colorectal cancer (CRC) cells. The evolutionarily conserved site K439 is identified as the key acetylation site. Tissue expression analysis demonstrates that the acetylation level of eEF1A1 K439 is higher thanpaired normal tissues. Most importantly, hyperacetylation of eEF1A1 at K439 negatively correlates with CRC patientsurvival. Mechanistically, CBP and SIRT1 are the majoracetyltransferase and deacetylase of eEF1A1. Hyperacetylation of eEF1A1 at K439 shows a significant tumor-promoting effect by increasing the capacity of proliferation, migration, and invasion of CRC cells. Our findings identify the altered post-translational modification at the translation machines as a critical factor instress response and susceptibility to colorectal carcinogenesis.

Structural changes of proteins in liver cirrhosis and consequential changes in their function.

The liver is the site of synthesis of the majority of circulating proteins. Besides initial polypeptide synthesis, sophisticated machinery is involved in the further processing of proteins by removing parts of them and/or adding functional groups and small molecules tailoring the final molecule to suit its physiological purpose. Posttranslational modifications (PTMs) design a network of molecules with the common protein ancestor but with slightly or considerably varying activity/localization/purpose. PTMs can change under pathological conditions, giving rise to aberrant or overmodified proteins. Undesired changes in the structure of proteins most often accompany undesired changes in their function, such as reduced activity or the appearance of new effects. Proper protein processing is essential for the reactions in living beings and crucial for the overall quality control. Modifications that occur on proteins synthesized in the liver whose PTMs are cirrhosis-related are oxidation, nitration, glycosylation, acetylation, and ubiquitination. Some of them predominantly affect proteins that remain in liver cells, whereas others predominantly occur on proteins that leave the liver or originate from other tissues and perform their function in the circulation. Altered PTMs of certain proteins are potential candidates as biomarkers of liver-related diseases, including cirrhosis. This review will focus on PTMs on proteins whose structural changes in cirrhosis exert or are suspected to exert the most serious functional consequences.

The Casein Kinase 2 Inhibitor CX-4945 Promotes Cholangiocarcinoma Cell Death Through PLK1.

Casein Kinase 2 (CK2) is a prosurvival protein kinase involved in cell growth/proliferation through the regulation of the cell cycle and apoptosis. CK2 is over-expressed in various cancers, which correlates with a poor prognosis. This study examined the anti-cancer effects of silmitasertib (CX-4945), a CK2 inhibitor, on cholangiocarcinoma (CCA) cells. The effects of CX-4945 on cell viability, cell cycle arrest, and apoptosis in the human cholangiocarcinoma cell lines TFK-1 and SSP-25 were evaluated. Alterations in posttranslational modifications and the levels of cell cycle regulators including p21, Polo-like kinase 1 (PLK1), andp53 were assessed by western blotting. Apoptotic responses were examined using Propidium iodine/Annexin V staining. TFK-1 and SSP-25 cells exposed to CX-4945 showed morphologic changes and a more than 50% decrease in cell viability (p<0.05). Cell cycle arrest at the G2 phase was detected following an increase in phosphorylated PLK1 and p21. Furthermore, phospho-PLK1 induced the degradation of p53, which led to the dissociation of Bax from Bcl-xL. The cleavage of Caspase3 and PARP were also induced by CX-4945 treatment. CX-4945 induces cell cycle arrest and cell death in cholangiocarcinoma cells via the regulation of PLK1 and p53. This may provide a novel therapeutic strategy for advanced cholangiocarcinoma.

Abstract 5027: PTMcosmos: A web portal of post-translational modifications and proteogenomic resources in cancer

Abstract PTMcosmos is a comprehensive database with an interactive web portal designed to catalog and visualize post-translational modifications (PTMs) in humans. It contains 469,183 experimentally-validated PTM sites and their supporting evidence from UniProt Knowledge Base, PhosphoSitePlus, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). PTMcosmos summarizes the entire spectrum of CPTAC proteomics data on human cancer patients, including protein and PTM peptide abundance data from 10 different cancer types. Additionally, PTMcosmos contains cancer somatic mutations from The Cancer Genome Atlas (TCGA), thus allowing for the collective integration and analysis of different data types. In PTMcosmos, we have built an ensemble of interactive visualization tools that allow researchers to investigate altered PTM functions due to genetic alterations in close proximity. The database is live at https://ptmcosmos.wustl.edu. We used PTMcosmos to investigate PTM regulation across cancer types. First, we examined the differential abundance of the PTM sites of cancer driver genes, focusing primarily on phosphorylation of the tumor suppressor retinoblastoma protein (encoded by RB1) and acetylation of the histone acetyltransferase E1A Binding Protein P300 (EP300) across cancer types. We analyzed the association between these PTM events and downstream targets, as well as with tumor subtypes, significantly mutated gene (SMG) mutation status, and clinical features. Second, we investigated the association of the protein abundance of cancer driver genes with ubiquitylsites in lung squamous cell carcinoma (LSCC) to nominate potentially novel modes of regulation of these proteins’ activities. We further analyzed the tumor subtype specificity and tumor-normal abundance changes of these ubiquitylsites and their corresponding substrate proteins, identifying several EGFR ubiquitylsites which may regulate EGFR abundance in LSCC. Finally, we identified the linear and spatial clustering of mutations and PTM sites, identifying multiple mutation-PTM clusters in cancer related genes, including TP53, PIK3CA, CTNNB1, EGFR, and IDH1. We envision that PTMcosmos will serve both the CPTAC consortium and the wider research community to better understand the role of PTMs in cancer. Citation Format: Liang-Bo Wang, Akshay Govindan, Song Cao, Li Ding. PTMcosmos: A web portal of post-translational modifications and proteogenomic resources in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5027.

The Role of Phytohormones in Plant Response to Flooding.

Climatic variations influence the morphological, physiological, biological, and biochemical states of plants. Plant responses to abiotic stress include biochemical adjustments, regulation of proteins, molecular mechanisms, and alteration of post-translational modifications, as well as signal transduction. Among the various abiotic stresses, flooding stress adversely affects the growth of plants, including various economically important crops. Biochemical and biological techniques, including proteomic techniques, provide a thorough understanding of the molecular mechanisms during flooding conditions. In particular, plants can cope with flooding conditions by embracing an orchestrated set of morphological adaptations and physiological adjustments that are regulated by an elaborate hormonal signaling network. With the help of these findings, the main objective is to identify plant responses to flooding and utilize that information for the development of flood-tolerant plants. This review provides an insight into the role of phytohormones in plant response mechanisms to flooding stress, as well as different mitigation strategies that can be successfully administered to improve plant growth during stress exposure. Ultimately, this review will expedite marker-assisted genetic enhancement studies in crops for developing high-yield lines or varieties with flood tolerance.

Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated invitro, invivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation invitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA invivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells invitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.

Oncohistones: a roadmap to stalled development.

Since the discovery of recurrent mutations in histone H3 variants in paediatric brain tumours, so-called 'oncohistones' have been identified in various cancers. While their mechanism of action remains under active investigation, several studies have shed light on how they promote genome-wide epigenetic perturbations. These findings converge on altered post-translational modifications on two key lysine (K) residues of the H3 tail, K27 and K36, which regulate several cellular processes, including those linked to cell differentiation during development. We will review how these oncohistones affect the methylation of cognate residues, but also disrupt the distribution of opposing chromatin marks, creating genome-wide epigenetic changes which participate in the oncogenic process. Ultimately, tumorigenesis is promoted through the maintenance of a progenitor state at the expense of differentiation in defined cellular and developmental contexts. As these epigenetic disruptions are reversible, improved understanding of oncohistone pathogenicity can result in needed alternative therapies.

A Comprehensive Analysis of the Role of hnRNP A1 Function and Dysfunction in the Pathogenesis of Neurodegenerative Disease.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a member of the hnRNP family of conserved proteins that is involved in RNA transcription, pre-mRNA splicing, mRNA transport, protein translation, microRNA processing, telomere maintenance and the regulation of transcription factor activity. HnRNP A1 is ubiquitously, yet differentially, expressed in many cell types, and due to post-translational modifications, can vary in its molecular function. While a plethora of knowledge is known about the function and dysfunction of hnRNP A1 in diseases other than neurodegenerative disease (e.g., cancer), numerous studies in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, multiple sclerosis, spinal muscular atrophy, Alzheimer’s disease, and Huntington’s disease have found that the dysregulation of hnRNP A1 may contribute to disease pathogenesis. How hnRNP A1 mechanistically contributes to these diseases, and whether mutations and/or altered post-translational modifications contribute to pathogenesis, however, is currently under investigation. The aim of this comprehensive review is to first describe the background of hnRNP A1, including its structure, biological functions in RNA metabolism and the post-translational modifications known to modify its function. With this knowledge, the review then describes the influence of hnRNP A1 in neurodegenerative disease, and how its dysfunction may contribute the pathogenesis.

Identification of a novel genetic locus associated with immune-mediated thrombotic thrombocytopenic purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare, life-threatening disorder, mediated through severe ADAMTS13 deficiency causing multi-system micro-thrombi formation, and has specific human leukocyte antigen associations. We undertook a large genome-wide association study to investigate additional genetically distinct associations in iTTP. We compared two iTTP patient cohorts with controls, following standardized genome-wide quality control procedures for single-nucleotide polymorphisms and imputed HLA types. Associations were functionally investigated using expression quantitative trait loci (eQTL), and motif binding prediction software. Independent associations consistent with previous findings in iTTP were detected at the HLA locus and in addition a novel association was detected on chromosome 3 (rs9884090, P=5.22x10-10, odds ratio 0.40) in the UK discovery cohort. Meta-analysis, including the French replication cohort, strengthened the associations. The haploblock containing rs9884090 is associated with reduced protein O-glycosyltransferase 1 (POGLUT1) expression (eQTL P<0.05), and functional annotation suggested a potential causative variant (rs71767581). This work implicates POGLUT1 in iTTP pathophysiology and suggests altered post-translational modification of its targets may influence disease susceptibility.

Systematic Proteome and Lysine Succinylome Analysis Reveals Enhanced Cell Migration by Hyposuccinylation in Esophageal Squamous Cell Carcinoma

AbstractEsophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and posttranslational modifications (PTMs) leading to the pathogenesis of ESCC remain unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome, and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and PTM pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

Ciliopathies are clinical disorders of the primary cilium with widely recognised phenotypic and genetic heterogeneity. Here, we found impaired ciliogenesis in fibroblasts derived from individuals with fetal akinesia deformation sequence (FADS), a broad spectrum of neuromuscular disorders arising from compromised foetal movement. We show that cells derived from FADS individuals have shorter and less primary cilia (PC), in association with alterations in post-translational modifications in α-tubulin. Similarly, siRNA-mediated depletion of two known FADS proteins, the scaffold protein rapsyn and the nucleoporin NUP88, resulted in defective PC formation. Consistent with a role in ciliogenesis, rapsyn and NUP88 localised to centrosomes and PC. Furthermore, proximity-ligation assays confirm the respective vicinity of rapsyn and NUP88 to γ-tubulin. Proximity-ligation assays moreover show that rapsyn and NUP88 are adjacent to each other and that the rapsyn-NUP88 interface is perturbed in the examined FADS cells. We suggest that the perturbed rapsyn-NUP88 interface leads to defects in PC formation and that defective ciliogenesis contributes to the pleiotropic defects seen in FADS.

GSK3β and Tau Protein in Alzheimer's Disease and Epilepsy.

Alzheimer’s disease (AD) is the most common form of dementia present in older adults; its etiology involves genetic and environmental factors. In recent years, epidemiological studies have shown a correlation between AD and chronic epilepsy since a considerable number of patients with AD may present seizures later on. Although the pathophysiology of seizures in AD is not completely understood, it could represent the result of several molecular mechanisms linked to amyloid beta-peptide (Aβ) accumulation and the hyperphosphorylation of tau protein, which may induce an imbalance in the release and recapture of excitatory and inhibitory neurotransmitters, structural alterations of the neuronal cytoskeleton, synaptic loss, and neuroinflammation. These changes could favor the recurrent development of hypersynchronous discharges and epileptogenesis, which, in a chronic state, favor the neurodegenerative process and influence the cognitive decline observed in AD. Supporting this correlation, histopathological studies in the brain tissue of temporal lobe epilepsy (TLE) patients have revealed the presence of Aβ deposits and the accumulation of tau protein in the neurofibrillary tangles (NFTs), accompanied by an increase of glycogen synthase kinase-3 beta (GSK3β) activity that may lead to an imminent alteration in posttranslational modifications of some microtubule-associated proteins (MAPs), mainly tau. The present review is focused on understanding the pathological aspects of GSK3β and tau in the development of TLE and AD.

Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.

Glycosylation influences activity, stability and immobilization of the feruloyl esterase 1a from Myceliophthora thermophila

Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.