Post-translational Control Research Articles

Post-Translational Modifications Control Phase Transitions of Tau

Post-Translational Modifications Control Phase Transitions of Tau

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

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

Post-translational control of B lineage commitment.

Post-translational control of B lineage commitment.

NaV1.1 contributes to the cell cycle of human mesenchymal stem cells by regulating AKT and CDK2.

Non-excitable cells express sodium voltage-gated channel alpha subunit 1 gene and protein (known as SCN1A and NaV1.1, respectively); however, the functions of NaV1.1 are unclear. In this study, we investigated the role of SCN1A and NaV1.1 in human mesenchymal stem cells (MSCs). We found that SCN1A was expressed in MSCs, and abundant expression of NaV1.1 was observed in the endoplasmic reticulum; however, this expression was not found to be related to Na+ currents. SCN1A-silencing reduced MSC proliferation and delayed the cell cycle in the S phase. SCN1A silencing also suppressed the protein levels of CDK2 and AKT (herein referring to total AKT), despite similar mRNA expression, and inhibited AKT phosphorylation in MSCs. A cycloheximide-chase assay showed that SCN1A-silencing induced CDK2 but not AKT protein degradation in MSCs. A proteolysis inhibition assay using epoxomicin, bafilomycin A1 and NH4Cl revealed that both the ubiquitin-proteasome system and the autophagy and endo-lysosome system were irrelevant to CDK2 and AKT protein reduction in SCN1A-silenced MSCs. The AKT inhibitor LY294002 did not affect the degradation and nuclear localization of CDK2 in MSCs. Likewise, the AKT activator SC79 did not attenuate the SCN1A-silencing effects on CDK2 in MSCs. These results suggest that NaV1.1 contributes to the cell cycle of MSCs by regulating the post-translational control of AKT and CDK2.

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells.

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure-activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Back to the Origin: Mechanisms of circRNA-Directed Regulation of Host Genes in Human Disease.

Circular RNAs (circRNAs) have been shown to be pivotal regulators in various human diseases by participating in gene splicing, acting as microRNA (miRNA) sponges, interacting with RNA-binding proteins (RBPs), and translating into short peptides. As the back-splicing products of pre-mRNAs, many circRNAs can modulate the expression of their host genes through transcriptional, post-transcriptional, translational, and post-translational control via interaction with other molecules. This review provides a detailed summary of these regulatory mechanisms based on the class of molecules that they interact with, which encompass DNA, mRNA, miRNA, and RBPs. The co-expression of circRNAs with their parental gene productions (including linear counterparts and proteins) provides potential diagnostic biomarkers for multiple diseases. Meanwhile, the different regulatory mechanisms by which circRNAs act on their host genes via interaction with other molecules constitute complex regulatory networks, which also provide noticeable clues for therapeutic strategies against diseases. Future research should explore whether these proven mechanisms can play a similar role in other types of disease and clarify further details about the cross-talk between circRNAs and host genes. In addition, the regulatory relationship between circRNAs and their host genes in circRNA circularization, degradation, and cellular localization should receive further attention.

A hierarchical ubiquitination-mediated regulatory module controls bamboo lignin biosynthesis

Abstract The lignocellulosic feedstock of woody bamboo shows promising potential as an alternative to conventional wood, attributed to its excellent properties. The content and distribution of lignin serve as the foundation of these properties. While the regulation of lignin biosynthesis in bamboo has been extensively studied at the transcriptional level, its posttranslational control has remained poorly understood. This study provides a ubiquitinome dataset for moso bamboo (Phyllostachys edulis), identifying 13015 ubiquitinated sites in 4849 unique proteins. We further identified Kelch repeat F-boxprotein 9 (PeKFB9) that plays a negative role in lignin biosynthesis. Heterologous expression of PeKFB9 resulted in reduced accumulation of lignin and decreased phenylalanine ammonia-lyase (PAL) activities. Both in vitro and in vivo assays identified interaction between PeKFB9 and PePAL10. Further examination revealed that SCFPeKFB9 mediated the ubiquitination and degradation of PePAL10 via the 26S proteasome pathway. Moreover, PebZIP28667 could bind to the PePAL10 promoter to significantly inhibit its transcription, and ubiquitination of PebZIP28667 weakened this inhibition. Collectively, our findings reveal a PeKFB9-PePAL10/PebZIP28667-PePAL10 module that acts as a negative regulator of lignin biosynthesis. This study advances our understanding of posttranslational regulation in plant lignification, which will facilitate the improvement of the properties of bamboo wood and the breeding of varieties.

PIN1 is a novel interaction partner and a negative upstream regulator of the transcription factor NFIB.

NFIB is a transcription factor of the Nuclear Factor One (NFI) family that is essential for embryonic development. Post-translational control of NFIB or its upstream regulators have not been well characterized. Here, we show that PIN1 binds NFIB in a phosphorylation-dependent manner, via its WW domain. PIN1 interacts with the well-conserved N-terminal domains of all NFIs. Moreover, PIN1 attenuates the transcriptional activity of NFIB; this attenuation requires substrate binding by PIN1 but not its isomerase activity. Paradoxically, we found stabilization of NFIB by PIN1. We propose that PIN1 represses NFIB function not by regulating its abundance but by inducing a conformational change. These results identify NFIB as a novel PIN1 target and posit a role for PIN1 in post-translational regulation of NFIB and other NFIs.

Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.

DCAF15 control of cohesin dynamics sustains acute myeloid leukemia

The CRL4-DCAF15 E3 ubiquitin ligase complex is targeted by the aryl-sulfonamide molecular glues, leading to neo-substrate recruitment, ubiquitination, and proteasomal degradation. However, the physiological function of DCAF15 remains unknown. Using a domain-focused genetic screening approach, we reveal DCAF15 as an acute myeloid leukemia (AML)-biased dependency. Loss of DCAF15 results in suppression of AML through compromised replication fork integrity and consequent accumulation of DNA damage. Accordingly, DCAF15 loss sensitizes AML to replication stress-inducing therapeutics. Mechanistically, we discover that DCAF15 directly interacts with the SMC1A protein of the cohesin complex and destabilizes the cohesin regulatory factors PDS5A and CDCA5. Loss of PDS5A and CDCA5 removal precludes cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication, and apoptosis. Collectively, our findings uncover an endogenous, cell autonomous function of DCAF15 in sustaining AML proliferation through post-translational control of cohesin dynamics.

Protein disulfide isomerase is essential for spermatogenesis in mice.

Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.

CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts.

The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.

O-GlcNAcylation Facilitates the Interaction between Keratin 18 and Isocitrate Dehydrogenases and Potentially Influencing Cholangiocarcinoma Progression.

Glycosylation plays a pivotal role in the intricate landscape of human cholangiocarcinoma (CCA), actively participating in key pathophysiological processes driving tumor progression. Among the various glycosylation modifications, O-linked β-N-acetyl-glucosamine modification (O-GlcNAcylation) emerges as a dynamic regulator influencing diverse tumor-associated biological activities. In this study, we employed a state-of-the-art chemical proteomic approach to analyze intact glycopeptides, unveiling the critical role of O-GlcNAcylation in orchestrating Keratin 18 (K18) and its interplay with tricarboxylic acid (TCA) cycle enzymes, specifically isocitrate dehydrogenases (IDHs), to propel CCA progression. Our findings shed light on the mechanistic intricacies of O-GlcNAcylation, revealing that site-specific modification of K18 on Ser 30 serves as a stabilizing factor, amplifying the expression of cell cycle checkpoints. This molecular event intricately fosters cell cycle progression and augments cellular growth in CCA. Notably, the interaction between O-GlcNAcylated K18 and IDHs orchestrates metabolic reprogramming by down-regulating citrate and isocitrate levels while elevating α-ketoglutarate (α-KG). These metabolic shifts further contribute to the overall tumorigenic potential of CCA. Our study thus expands the current understanding of protein O-GlcNAcylation and introduces a new layer of complexity to post-translational control over metabolism and tumorigenesis.

KIS counteracts PTBP2 and regulates alternative exon usage in neurons.

Alternative RNA splicing is an essential and dynamic process in neuronal differentiation and synapse maturation, and dysregulation of this process has been associated with neurodegenerative diseases. Recent studies have revealed the importance of RNA-binding proteins in the regulation of neuronal splicing programs. However, the molecular mechanisms involved in the control of these splicing regulators are still unclear. Here, we show that KIS, a kinase upregulated in the developmental brain, imposes a genome-wide alteration in exon usage during neuronal differentiation in mice. KIS contains a protein-recognition domain common to spliceosomal components and phosphorylates PTBP2, counteracting the role of this splicing factor in exon exclusion. At the molecular level, phosphorylation of unstructured domains within PTBP2 causes its dissociation from two co-regulators, Matrin3 and hnRNPM, and hinders the RNA-binding capability of the complex. Furthermore, KIS and PTBP2 display strong and opposing functional interactions in synaptic spine emergence and maturation. Taken together, our data uncover a post-translational control of splicing regulators that link transcriptional and alternative exon usage programs in neuronal development.

Abstract 6321: Developing a lenalidomide-inducible safety switch for CAR T cell therapy

Abstract While CAR T cell therapy is a medical breakthrough against various hematological malignancies, yet treatment failures often occur at least in part due to excessive engineered T cell activity leading to severe toxicities. To combat this, strategies pairing CAR T cells with safety systems, for example, suicide switches engineered to induce cell death are being actively pursued. However, existing designs have limited performance characteristics and generally use unapproved small molecule controllers or non-human sequences. To overcome these issues, we have developed a novel suicide switch leveraging the well-tolerated, FDA-approved anti-cancer drug lenalidomide, as a controller of the stability of fusion proteins tagged with a “degron” sequence. Specifically, we engineered a stoichiometric pair of transgenes using the apoptotic DNase pair, caspase activating DNase (CAD) and its degron tagged inhibitor (ICAD-degron). We hypothesized that the overexpressed ICAD-degron could restrain CAD, until lenalidomide treatment degrades ICAD to unleash CAD for apoptosis. We found that our system was stably overexpressed and rapidly induced cell death even at sub-nanomolar concentrations of lenalidomide. A comparison of our design with the frontline safety switch, inducible Caspase 9 in primary human T cells revealed that the lenalidomide-induced suicide switch enabled more complete depletion of engineered cells. We tested the effects of our suicide switch on CAR T cell function and found comparable proliferation and tumor cell cytolysis with fast depletion upon lenalidomide addition. In vivo, lenalidomide suicide switch CAR T cells exhibited similar anti-tumor function as control CAR T cells and were rapidly depleted after drug treatment. In summary, utilizing all human sequences and a clinically approved drug controller, we developed a suicide switch that is well tolerated and can rapidly induce cell death even at sub-therapeutic concentrations of lenalidomide. More broadly, chemogenetic regulation of stoichiometric protein pairs is a generalizable strategy for post-translational control of highly active transgenic elements. Citation Format: Ditsa Sarkar, William Lin, Joanna Y. Kim, Nelson H. Knudsen, Isabel C. Lane, Tamina Kienka, Michael C. Kann, Amanda A. Bouffard, Andy Cheng, Marcela V. Maus, Robert T. Manguso, Max Jan. Developing a lenalidomide-inducible safety switch for CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6321.

Light-inducible protein degradation in E. coli with the LOVdeg tag.

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system and introduce a powerful new tool for bacterial optogenetics.

Light-inducible protein degradation in E. coli with the LOVdeg tag

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system and introduce a powerful new tool for bacterial optogenetics.

Late-Stage Functionalization of Living Organisms: Rethinking Selectivity in Biology.

With unlimited selectivity, full post-translational chemical control of biology would circumvent the dogma of genetic control. The resulting direct manipulation of organisms would enable atomic-level precision in "editing" of function. We argue that a key aspect that is still missing in our ability to do this (at least with a high degree of control) is the selectivity of a given chemical reaction in a living organism. In this Review, we systematize existing illustrative examples of chemical selectivity, as well as identify needed chemical selectivities set in a hierarchy of anatomical complexity: organismo- (selectivity for a given organism over another), tissuo- (selectivity for a given tissue type in a living organism), cellulo- (selectivity for a given cell type in an organism or tissue), and organelloselectivity (selectivity for a given organelle or discrete body within a cell). Finally, we analyze more traditional concepts such as regio-, chemo-, and stereoselective reactions where additionally appropriate. This survey of late-stage biomolecule methods emphasizes, where possible, functional consequences (i.e., biological function). In this way, we explore a concept of late-stage functionalization of living organisms (where "late" is taken to mean at a given state of an organism in time) in which programmed and selective chemical reactions take place in life. By building on precisely analyzed notions (e.g., mechanism and selectivity) we believe that the logic of chemical methodology might ultimately be applied to increasingly complex molecular constructs in biology. This could allow principles developed at the simple, small-molecule level to progress hierarchically even to manipulation of physiology.

Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase

Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and non-sterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. We infer from these results that, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.

Posttranslational Remote Control Mediated by Physical Inducers for Rapid Protein Release in Engineered Mammalian Cells.

Physical cues such as light, heat, or an electrical field can be utilized for traceless, on-demand activation of the expression of a desired therapeutic gene in appropriately engineered cells with excellent spatiotemporal resolution, good inducibility, and simple reversibility. A similar approach can be applied to build a depolarization-based protein secretion system that enables rapid release of a therapeutic protein pre-stored in intracellular vesicles in mammalian cells. Here, we present a protocol to create designer β-cells that exhibit light-controllable rapid release (within 15min) of a pre-synthesized proinsulin-nanoluciferase construct from vesicular stores. The construct is cleaved extracellularly to afford secreted insulin as a therapeutic protein and nanoluciferase as a reporter molecule. Such posttranslational remote control offers a much faster response than expression-based systems.