Post-translational Control Research Articles

One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines.

Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.

ACONITASE 3 is part of theANAC017 transcription factor-dependent mitochondrial dysfunction response.

Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Posttranslational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B’γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3S91D-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against ultraviolet B (UV-B) or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.

Proteomics and expression studies on lipids and fatty acids metabolic genes in Isochrysis galbana under the combined influence of nitrogen starvation and sodium acetate supplementation

The marine alga Isochrysis galbana was grown under the integrated influence of nitrogen starvation and sodium acetate, specifically to understand the associated remodelling of the fatty acid profiles. This is the first comparative proteomics study under four different set of conditions namely nutrient sufficient, with/without sodium acetate and N-deficient with/without sodium acetate. A total set of 546 unique peptides were detected. Due to the addition of sodium acetate, 56 proteins were down-regulated and 7 proteins showed up-regulation respectively in the nutrient complete and the nitrogen starvation media. Both cell growth and docosahexaenoic acid (DHA) accumulation in the nutrient replete cultures were favoured by the addition of reduced carbon source in the form of sodium acetate. The 14 days old sodium acetate supplemented nitrogen starved cells showed highest amounts of total fatty acids and an over accumulation of the oleic acid. Of the seven key genes of the lipid and fatty acid metabolic pathway that were studied, the IgTeCe, IgFad12, and IgFad4 showed over expressions under nitrogen starvation. The gene expressions of lipases and desaturases involved in the fatty acids metabolism were not found to be correlated with the changes observed in the fatty acid profiles under the experimental conditions. This suggested the presence of an alternative metabolic route and/or possibly the variants of fatty acid desaturase genes in I. galbana which was beyond the scope of this study. A translational or post-translational control can not either be ignored.

Post-translational Control of RNA-Binding Proteins and Disease-Related Dysregulation.

Cell signaling mechanisms modulate gene expression in response to internal and external stimuli. Cellular adaptation requires a precise and coordinated regulation of the transcription and translation processes. The post-transcriptional control of mRNA metabolism is mediated by the so-called RNA-binding proteins (RBPs), which assemble with specific transcripts forming messenger ribonucleoprotein particles of highly dynamic composition. RBPs constitute a class of trans-acting regulatory proteins with affinity for certain consensus elements present in mRNA molecules. However, these regulators are subjected to post-translational modifications (PTMs) that constantly adjust their activity to maintain cell homeostasis. PTMs can dramatically change the subcellular localization, the binding affinity for RNA and protein partners, and the turnover rate of RBPs. Moreover, the ability of many RBPs to undergo phase transition and/or their recruitment to previously formed membrane-less organelles, such as stress granules, is also regulated by specific PTMs. Interestingly, the dysregulation of PTMs in RBPs has been associated with the pathophysiology of many different diseases. Abnormal PTM patterns can lead to the distortion of the physiological role of RBPs due to mislocalization, loss or gain of function, and/or accelerated or disrupted degradation. This Mini Review offers a broad overview of the post-translational regulation of selected RBPs and the involvement of their dysregulation in neurodegenerative disorders, cancer and other pathologies.

Posttranslational and Therapeutic Control of Gasdermin-Mediated Pyroptosis and Inflammation.

Pyroptosis is a proinflammatory form of cell death, mediated by membrane pore-forming proteins called gasdermins. Gasdermin pores allow the release of the pro-inflammatory cytokines IL-1β and IL-18 and cause cell swelling and cell lysis leading to release of other intracellular proteins that act as alarmins to perpetuate inflammation. The best characterized, gasdermin D, forms pores via its N-terminal domain, generated after the cleavage of full length gasdermin D by caspase-1 or -11 (caspase-4/5 in humans) typically upon sensing of intracellular pathogens. Thus, gasdermins were originally thought to largely contribute to pathogen-induced inflammation. We now know that gasdermin family members can also be cleaved by other proteases, such as caspase-3, caspase-8 and granzymes, and that they contribute to sterile inflammation as well as inflammation in autoinflammatory diseases or during cancer immunotherapy. Here we briefly review how and when gasdermin pores are formed, and then focus on emerging endogenous mechanisms and therapeutic approaches that could be used to control pore formation, pyroptosis and downstream inflammation.

Storage time and temperature affects volatile organic compound profile, alliinase activity and postharvest quality of garlic

Garlic (Allium sativum L.) has a long history of use as a culinary seasoning and source of health-promoting compounds. In particular organosulphur compounds derived from the action of alliinase on alliin are of interest for their antimicrobial action. Due to the seasonal nature of the garlic harvest, long-term storage is required to ensure year-round supply of high-quality bulbs. However, quality is known to deteriorate throughout storage, and storage regimes are aimed at maintaining culinary, not biochemical quality, posing challenges for biotech firms extracting high value products, such as alliinase, from garlic. Storage typically involves extended periods of up to 9 months at -1.5 °C. Here, quality parameters (disease incidence and moisture content) as well as biotechnological quality parameters (alliinase yield and activity) were measured, and correlated with gene expression and volatile organic compound (VOC) profiles comparing storage at -1.5 °C and 22 °C. The aim is to develop potential molecular markers for garlic quality assessment. Alliinase activity fell in the first 6 months of storage, with garlic stored at -1.5 °C losing more activity, however 22 °C stored garlic suffered higher spoilage after 12 months storage. Alliinase activity loss was not proportional to gene transcript levels, suggesting post-translational control. A total of 150 VOCs were detected across all samples using thermal desorption gas chromatography, time of flight mass spectrometry of intact garlic bulbs, the most abundant of which were organosulphur compounds. Storage temperature significantly affected the whole VOC profile and discrete profiles were detected from garlic cold-stored for different time periods. Using weighted correlation network analysis 17 VOCs were identified that correlated with storage time, six VOCs that were indicative for storage temperature and four VOCs (azulene, octanal, o-Xylene and 4-methylhexadecane) were significantly associated with alliinase activity.

Phytochrome Signaling Networks.

The perception of light signals by the phytochrome family of photoreceptors has a crucial influence on almost all aspects of growth and development throughout a plant's life cycle. The holistic regulatory networks orchestrated by phytochromes, including conformational switching, subcellular localization, direct protein-protein interactions, transcriptional and posttranscriptional regulations, and translational and posttranslational controls to promote photomorphogenesis, are highly coordinated and regulated at multiple levels. During the past decade, advances using innovative approaches have substantially broadened our understanding of the sophisticated mechanisms underlying the phytochrome-mediated light signaling pathways. This review discusses and summarizes these discoveries of the role of the modular structure of phytochromes, phytochrome-interacting proteins, and their functions; the reciprocal modulation of both positive and negative regulators in phytochrome signaling; the regulatory roles of phytochromes in transcriptional activities, alternative splicing, and translational regulation; and the kinases and E3 ligases that modulate PHYTOCHROME INTERACTING FACTORs to optimize photomorphogenesis.

Effects of Resistance Training on the Redox Status of Skeletal Muscle in Older Adults.

The aim of this study was to investigate the effects of resistance training (RT) on the redox status of skeletal muscle in older adults. Thirteen males aged 64 ± 9 years performed full-body RT 2x/week for 6 weeks. Muscle biopsies were obtained from the vastus lateralis prior to and following RT. The mRNA, protein, and enzymatic activity levels of various endogenous antioxidants were determined. In addition, skeletal muscle 4-hydroxynonenal and protein carbonyls were determined as markers of oxidative damage. Protein levels of heat shock proteins (HSPs) were also quantified. RT increased mRNA levels of all assayed antioxidant genes, albeit protein levels either did not change or decreased. RT increased total antioxidant capacity, catalase, and glutathione reductase activities, and decreased glutathione peroxidase activity. Lipid peroxidation also decreased and HSP60 protein increased following RT. In summary, 6 weeks of RT decreased oxidative damage and increased antioxidant enzyme activities. Our results suggest the older adult responses to RT involve multi-level (transcriptional, post-transcriptional, and post-translational) control of the redox status of skeletal muscle.

Model-guided design of mammalian genetic programs.

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

A platform for post-translational spatiotemporal control of cellular proteins.

Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.

Post-translational modification control of viral DNA sensors and innate immune signaling.

The vertebrate innate immune system confers host cells with mechanisms to protect against both evolutionarily ancient pathogens and newly emerging pathogenic strains. Innate immunity relies on the host cell's ability to distinguish between self and pathogen-derived molecules. To achieve this, the innate immune system uses germline encoded receptors called pattern recognition receptors (PRRs), which recognize various molecular signatures, including nucleic acids, proteins, lipids, glycans and glycolipids. Among these molecules, the recognition of pathogenic, mislocalized, or damaged DNA by cellular protein receptors, commonly called DNA sensors, represents a major surveillance pathway for initiating immune signaling. The ability of cells to temporally regulate DNA sensor activation and subsequent signal termination is critical for effective immune signaling. These same mechanisms are also co-opted by pathogens to promote their replication. Therefore, there is significant interest in understanding DNA sensor regulatory networks during microbial infections and autoimmune disease. One emerging aspect of DNA sensor regulation is through post-translational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, ADP-ribosylation, SUMOylation, methylation, deamidation, glutamylation. In this chapter, we discuss how PTMs have been shown to positively or negatively impact DNA sensor functions via diverse mechanisms, including direct regulation of enzymatic activity, protein-protein and protein-DNA interactions, protein translocations and protein turnover. In addition, we highlight the ability of virus-induced PTMs to promote immune evasion. We also discuss the recent evidence linking PTMs on DNA sensors with human diseases and more broadly, highlight promising directions for future research on PTM-mediated regulation of DNA sensor-dependent immune signaling.

The Conditional Knockout Analogous System: CRISPR-Mediated Knockout Together with Inducible Degron and Transcription-Controlled Expression.

The revolutionary CRISPR technology opens a new era of cell biology in mammalian cells. The InDel mutation is induced by CRISPR and results in the frameshift mutation of the gene. Owing to the nature of CRISPR induced knockout, the conditional knockout using CRISPR technology is not common. With the recent development of the small molecule-inducible degron system, an analogous system to the classical genetic conditional knockout has become feasible. By integrating CRISPR-knockout, the tetracycline-controlled transcriptional and auxin-induced degradation post-translational control of protein expression, a method imitating the conditional knockout is developed. We herein describe the detailed protocol for the generation of a conditional protein inactivation in human cancer cells. The system is especially useful to study essential gene function in aneuploidy cancer cells where gain in copy number is common.

In vitro Fluid Shear Stress Induced Sclerostin Degradation and CaMKII Activation in Osteocytes.

Bone is a dynamic tissue that adapts to changes in its mechanical environment. Mechanical stimuli pressurize interstitial fluid in the lacunar-canalicular system within the bone matrix, causing fluid shear stress (FSS) across bone embedded, mechano-sensitive osteocytes. Therefore, modeling this mechanical stimulus in vitro is vital for identifying mechano-transduction cascades that contribute to the regulation of mechano-responsive proteins, such as the Wnt/β-catenin antagonist, sclerostin, which is reduced in response to FSS. Recently, we reported the rapid post-translational degradation of sclerostin protein in bone cells following FSS. Given the fundamental nature of sclerostin to bone physiology and the nuances of studying its rapid post-translational control, here, we detail our FSS protocol, and adaptations that can be made, to stimulate Ocy454 osteocyte-like cells to study sclerostin protein in vitro. While this protocol is optimized for detecting sclerostin degradation by western blot, this protocol can be adapted to examine transcriptional changes with RT-qPCR, cellular dynamics with live cell imaging, or secreted factors in the FSS buffer. This protocol utilizes 3D-printed FSS tips that are compatible with commercially available 96-well plates, allowing for high experimental accessibility, versatility, and throughput. However, this protocol can be adapted for any FSS chamber. It can also be combined with pharmacological inhibitors or genetic manipulations to interrogate the role of specific cellular components. In all, this experimental set-up and protocol is highly adaptable to allow for many experimental outcomes to examine many aspects of cell mechano-transduction.

Transcriptional and Post-Translational Control Mechanisms for EphB4 Expression in Physiology and Cancer Disease.

EphB4 is a membrane tyrosine kinase receptor involved in a number of physiologic and pathologic conditions, cancer being the one drawing the highest attention. In mammalians, EphB4 regulates blood vessel growth and differentiation. EphB4 also mediates a number of cancer promoting effects including angiogenesis, invasion, and metastasis. Its effects have been linked to both EphrinB2-dependent and to EphrinB2-independent mechanisms. In spite of its emerging role in physiology and disease, the regulation of EphB4 cellular expression has been poorly described until recently. The present mini-review summarizes the latest findings on EphB4 regulation at the transcriptional and post-transcriptional level and discusses the biologic implications of such novel findings. Perspective on the scenarios opened by such findings and the open questions in EphB4 research is also provided.

ELK-1 ubiquitination status and transcriptional activity are modulated independently of F-Box protein FBXO25

The mitogen-responsive, ETS-domain transcription factor ELK-1 stimulates the expression of immediate early genes at the onset of the cell cycle and participates in early developmental programming. ELK-1 is subject to multiple levels of posttranslational control, including phosphorylation, SUMOylation, and ubiquitination. Recently, removal of monoubiquitin from the ELK-1 ETS domain by the Ubiquitin Specific Protease USP17 was shown to augment ELK-1 transcriptional activity and promote cell proliferation. Here we have used coimmunoprecipitation experiments, protein turnover and ubiquitination assays, RNA-interference and gene expression analyses to examine the possibility that USP17 acts antagonistically with the F-box protein FBXO25, an E3 ubiquitin ligase previously shown to promote ELK-1 ubiquitination and degradation. Our data confirm that FBXO25 and ELK-1 interact in HEK293T cells and that FBXO25 is active toward Hand1 and HAX1, two of its other candidate substrates. However, our data indicate that FBXO25 neither promotes ubiquitination of ELK-1 nor impacts on its transcriptional activity and suggest that an E3 ubiquitin ligase other than FBXO25 regulates ELK-1 ubiquitination and function.

Article of Significant Interest in This Issue

In bacteria, the Lon protease destroys poor-quality polypeptides to prevent the toxic accumulation of misfolded proteins. However, Lon also degrades essential regulatory proteins, such as DnaA in Caulobacter crescentus; hence, activity must be carefully monitored. Barros et al. (e00344-20) show that the Lon protease is itself degraded in vivo. This process requires active Lon proteases and requires carboxy-terminal residues also found in various Lon orthologs. In Caulobacter, expression of stabilized Lon drastically reduces DnaA levels, leading to cell death. They suggest that this posttranslational control balances the requirement for Lon with the toxicity associated with unconstrained proteolysis.

Protein phosphatase NtPP2C2b and MAP kinase NtMPK4 act in concert to modulate nicotine biosynthesis.

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

Allomorphy as a mechanism of post-translational control of enzyme activity

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Regulation of ABCG4 transporter expression by sterols and LXR ligands

BackgroundOxysterols, which are derivatives of cholesterol produced by enzymic or non-enzymic pathways, are potent regulators of cellular lipid homeostasis. Sterol homeostasis in the brain is an important area of interest with regards to neurodegenerative conditions like Alzheimer's disease (AD). Brain cells including neurons and astrocytes express sterol transporters belonging to the ABC transporter family of proteins, including ABCA1, ABCG1 and ABCG4, and these transporters are considered of interest as therapeutic targets. Although regulation of ABCA1 and ABCG1 is well established, regulation of ABCG4 is still controversial, in particular whether the transporter is an Liver X receptor (LXR) target. ABCG4 is thought to transport cholesterol, oxysterols and cholesterol synthesis intermediates, and was recently found on the blood brain barrier (BBB), implicated in amyloid-beta export. In this study, we investigate the regulation of ABCG4 by oxysterols, cholesterol-synthesis intermediates and cholesterol itself. MethodsABC transporter expression was measured in neuroblastoma and gliablastoma cell lines and cells overexpressing ABCG4 in response to synthetic LXR ligands, oxysterols and cholesterol-synthesis intermediates. ResultsIn contrast to previous reports, ABCG4 expression was induced by a synthetic LXR ligand in U87-MG astrocytes but not in neuroblastoma and BBB endothelial cell lines. In addition, ABCG4 protein was stabilized by cholesterol as was previously shown for ABCG1. ABCG4 protein was furthermore stabilized by cholesterol-synthesis intermediates, desmosterol, lathosterol and lanosterol. ConclusionsThese results identify new aspects of the post-translational control of ABCG4 that warrant further exploration into the role of this transporter in the maintenance of sterol homeostasis in the brain.