A Conserved Mechanism of Cardiac Hypertrophy Regression through FoxO1.

The heart is a highly plastic organ that responds to diverse stimuli to modify form and function. The molecular mechanisms of adaptive physiological cardiac hypertrophy are well-established; however, the regulation of hypertrophy regression is poorly understood. To identify molecular features of regression, we studied Burmese pythons which experience reversible cardiac hypertrophy following large, infrequent meals. Using multi-omics screens followed by targeted analyses, we found forkhead box protein O1 (FoxO1) transcription factor signaling, and downstream autophagy activity, were downregulated during hypertrophy, but re-activated with regression. To determine whether these events were mechanistically related to regression, we established an in vitro platform of cardiomyocyte hypertrophy and regression from treatment with fed python plasma. FoxO1 inhibition prevented regression in this system, while FoxO1 activation reversed fed python plasma-induced hypertrophy in an autophagy-dependent manner. We next examined whether FoxO1 was implicated in mammalian models of reversible hypertrophy from exercise and pregnancy and found that in both cases FoxO1 was activated during regression. In these models, as in pythons, activation of FoxO1 was associated with increased expression FoxO1 target genes involved in autophagy. Taken together, our findings suggest FoxO1-dependent autophagy is a conserved mechanism for regression of physiological cardiac hypertrophy across species.

Cooperative polarization of MCAM/CD146 and ERM family proteins in melanoma.

The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile ("3P"), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although copolarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.

Simultaneous CAS9 editing of cpSRP43, LHCA6, and LHCA7 in Picochlorum celeri lowers chlorophyll levels and improves biomass productivity.

High cellular pigment levels in dense microalgal cultures contribute to excess light absorption. To improve photosynthetic yields in the marine microalga Picochlorum celeri, CAS9 gene editing was used to target the molecular chaperone cpSRP43. Depigmented strains (>50% lower chlorophyll) were generated, with proteomics showing attenuated levels of most light harvesting complex (LHC) proteins. Gene editing generated two types of cpSRP43 transformants with distinct lower pigment phenotypes: (i) a transformant (Δsrp43) with both cpSRP43 diploid alleles modified to encode non-functional polypeptides and (ii) a transformant (STR30309) with a 3 nt in-frame insertion in one allele at the CAS9 cut site (non-functional second allele), leading to expression of a modified cpSRP43. STR30309 has more chlorophyll than Δsrp43 but substantially less than wild type. To further decrease light absorption by photosystem I in STR30309, CAS9 editing was used to stack in disruptions of both LHCA6 and LHCA7 to generate STR30843, which has higher (5-24%) productivities relative to wild type in solar-simulating bioreactors. Maximal productivities required frequent partial harvests throughout the day. For STR30843, exemplary diel bioreactor yields of ~50 g m-2 day-1 were attained. Our results demonstrate diel productivity gains in P. celeri by lowering pigment levels.

The identification of protein and RNA interactors of the splicing factor Caper in the adult Drosophila nervous system.

Post-transcriptional gene regulation is a fundamental mechanism that helps regulate the development and healthy aging of the nervous system. Mutations that disrupt the function of RNA-binding proteins (RBPs), which regulate post-transcriptional gene regulation, have increasingly been implicated in neurological disorders including amyotrophic lateral sclerosis, Fragile X Syndrome, and spinal muscular atrophy. Interestingly, although the majority of RBPs are expressed widely within diverse tissue types, the nervous system is often particularly sensitive to their dysfunction. It is therefore critical to elucidate how aberrant RNA regulation that results from the dysfunction of ubiquitously expressed RBPs leads to tissue specific pathologies that underlie neurological diseases. The highly conserved RBP and alternative splicing factor Caper is widely expressed throughout development and is required for the development of Drosophila sensory and motor neurons. Furthermore, caper dysfunction results in larval and adult locomotor deficits. Nonetheless, little is known about which proteins interact with Caper, and which RNAs are regulated by Caper. Here we identify proteins that interact with Caper in both neural and muscle tissue, along with neural specific Caper target RNAs. Furthermore, we show that a subset of these Caper-interacting proteins and RNAs genetically interact with caper to regulate Drosophila gravitaxis behavior.

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS cases are caused by mutation of the proteasome shuttle factor Ubiquilin 2 (UBQLN2), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate that UBQLN2 regulates the domesticated gag-pol retrotransposon 'paternally expressed gene 10 (PEG10)' in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated 'nucleocapsid' fragment, which uniquely localizes to the nucleus and changes the expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through the regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

Molecular insight into the specific interactions of the SARS-Coronavirus-2 nucleocapsid with RNA and host protein.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid protein is the most abundantly expressed viral protein during infection where it targets both RNA and host proteins. However, identifying how a single viral protein interacts with so many different targets remains a challenge, providing the impetus here for identifying the interaction sites through multiple methods. Through a combination of nuclear magnetic resonance (NMR), electron microscopy, and biochemical methods, we have characterized nucleocapsid interactions with RNA and with three host proteins, which include human cyclophilin-A, Pin1, and 14-3-3τ. Regarding RNA interactions, the nucleocapsid protein N-terminal folded domain preferentially interacts with smaller RNA fragments relative to the C-terminal region, suggesting an initial RNA engagement is largely dictated by this N-terminal region followed by weaker interactions to the C-terminal region. The nucleocapsid protein forms 10 nm ribonuclear complexes with larger RNA fragments that include 200 and 354 nucleic acids, revealing its potential diversity in sequestering different viral genomic regions during viral packaging. Regarding host protein interactions, while the nucleocapsid targets all three host proteins through its serine-arginine-rich region, unstructured termini of the nucleocapsid protein also engage host cyclophilin-A and host 14-3-3τ. Considering these host proteins play roles in innate immunity, the SARS-CoV-2 nucleocapsid protein may block the host response by competing interactions. Finally, phosphorylation of the nucleocapsid protein quenches an inherent dynamic exchange process within its serine-arginine-rich region. Our studies identify many of the diverse interactions that may be important for SARS-CoV-2 pathology during infection.

MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.

Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.

Systematic mutagenesis of TFIIH subunit p52/Tfb2 identifies residues required for XPB/Ssl2 subunit function and genetic interactions with TFB6.

TFIIH is an evolutionarily conserved complex that plays central roles in both RNA polymerase II (pol II) transcription and DNA repair. As an integral component of the pol II preinitiation complex, TFIIH regulates pol II enzyme activity in numerous ways. The TFIIH subunit XPB/Ssl2 is an ATP-dependent DNA translocase that stimulates promoter opening prior to transcription initiation. Crosslinking-mass spectrometry and cryo-EM results have shown a conserved interaction network involving XPB/Ssl2 and the C-terminal Hub region of the TFIIH p52/Tfb2 subunit, but the functional significance of specific residues is unclear. Here, we systematically mutagenized the HubA region of Tfb2 and screened for growth phenotypes in a TFB6 deletion background in Saccharomyces cerevisiae. We identified six lethal and 12 conditional mutants. Slow growth phenotypes of all but three conditional mutants were relieved in the presence of TFB6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Our biochemical analysis of Tfb2 mutants with severe growth phenotypes revealed defects in Ssl2 association, with similar results in human cells. Further characterization of these tfb2 mutant cells revealed defects in GAL gene induction, and reduced occupancy of TFIIH and pol II at GAL gene promoters, suggesting that functionally competent TFIIH is required for proper pol II recruitment to preinitiation complexes in vivo. Consistent with recent structural models of TFIIH, our results identify key residues in the p52/Tfb2 HubA domain that are required for stable incorporation of XPB/Ssl2 into TFIIH and for pol II transcription.

Taf2 mediates DNA binding of Taf14.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited. Binding of Taf2 promotes a conformational rearrangement in Taf14, resulting in a release of the linker for the engagement with DNA and the nucleosome. Genetic in vivo data indicate that the association of Taf14 with both Taf2 and DNA is essential for transcriptional regulation. Our findings provide a basis for deciphering the role of individual TFIID subunits in mediating gene transcription.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide.

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this "bivalent" peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs.

The TORC1 phosphoproteome in C. elegans reveals roles in transcription and autophagy.

The protein kinase complex target of rapamycin complex 1 (TORC1) is a critical mediator of nutrient sensing that has been widely studied in cultured cells and yeast, yet our understanding of the regulatory activities of TORC1 in the context of a whole, multi-cellular organism is still very limited. Using Caenorhabditis elegans, we analyzed the DAF-15/Raptor-dependent phosphoproteome by quantitative mass spectrometry and characterized direct kinase targets by in vitro kinase assays. Here, we show new targets of TORC1 that indicate previously unknown regulation of transcription and autophagy. Our results further show that DAF-15/Raptor is differentially expressed during postembryonic development, suggesting a dynamic role for TORC1 signaling throughout the life span. This study provides a comprehensive view of the TORC1 phosphoproteome, reveals more than 100 DAF-15/Raptor-dependent phosphosites that reflect the complex function of TORC1 in a whole, multi-cellular organism, and serves as a rich resource to the field.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs.

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity.

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Changes to the TDP-43 and FUS Interactomes Induced by DNA Damage.

The RNA-binding proteins TDP-43 and FUS are tied as the third leading known genetic cause for amyotrophic lateral sclerosis (ALS), and TDP-43 proteopathies are found in nearly all ALS patients. Both the natural function and contribution to pathology for TDP-43 remain unclear. The intersection of functions between TDP-43 and FUS can focus attention for those natural functions mostly likely to be relevant to disease. Here, we compare the role played by TDP-43 and FUS, maintaining chromatin stability for dividing HEK293T cells. We also determine and compare the interactomes of TDP-43 and FUS, quantitating changes in those before and after DNA damage. Finally, selected interactions with known importance to DNA damage repair were validated by co-immunoprecipitation assays. This study uncovered TDP-43 and FUS binding to several factors important to DNA repair mechanisms that can be replication-dependent, -independent, or both. These results provide further evidence that TDP-43 has an important role in DNA stability and provide new ways that TDP-43 can bind to the machinery that guards DNA integrity in cells.

Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome.

New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the 'weak-link' enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.

Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB.

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP) in Escherichia coli Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.