Mst4, a novel cardiac STRIPAK complex-associated kinase, regulates cardiomyocyte growth and survival and is upregulated in human cardiomyopathy.

Myocardial failure is associated with adverse remodeling, including loss of cardiomyocytes, hypertrophy, and alterations in cell-cell contacts. Striatin-interacting phosphatase and kinase (STRIPAK) complexes and their mammalian STE20-like kinase 4 (Mst4) have been linked to development of different diseases. The role and targets of Mst4 in cardiomyocytes have not been investigated yet. Multitissue immunoblot experiments show highly enriched Mst4 expression in rodent hearts. Analyses of human biopsy samples from patients suffering from dilated cardiomyopathy revealed that Mst4 is upregulated (5- to 8-fold p < 0.001) compared with nonfailing controls. Increased abundance of Mst4 could also be detected in mouse models of cardiomyopathy. We confirmed that Mst4 interacts with STRIPAK components in neonatal rat ventricular cardiomyocytes, indicating that STRIPAK is present in the heart. Immunofluorescence stainings and molecular interaction studies revealed that Mst4 is localized to the intercalated disc and interacts with several intercalated disc proteins. Overexpression of Mst4 in cardiomyocytes results in hypertrophy compared with controls. In adult rat cardiomyocytes, Mst4 overexpression increases cellular and sarcomeric fractional shortening (p < 0.05), indicating enhanced contractility. Overexpression of Mst4 also inhibits apoptosis shown by reduction of cleaved caspase3 (-69%, p < 0.0001), caspase7 (-80%, p < 0.0001), and cleaved Parp1 (-27%, p < 0.001). To elucidate potential Mst4 targets, we performed phosphoproteomics analyses in neonatal rat cardiomyocytes after Mst4 overexpression and inhibition. The results revealed target candidates of Mst4 at the intercalated disc. We identified Mst4 as a novel cardiac kinase that is upregulated in cardiomyopathy-regulating cardiomyocyte growth and survival.

Synchronized assembly of the oxidative phosphorylation system controls mitochondrial respiration in yeast.

Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the ratio of the electron transport chain (ETC) and ATP synthase must be tightly controlled, and assembly requires coordinated integration of proteins encoded in the nuclear and mitochondrial genome. How correct OXPHOS stoichiometry is achieved is unknown. We identify the Mitochondrial Regulatory hub for respiratory Assembly (MiRA) platform, which synchronizes ETC and ATP synthase biogenesis in yeast. Molecularly, this is achieved by a stop-and-go mechanism: the uncharacterized protein Mra1 stalls complex IV assembly. Two "Go" signals are required for assembly progression: binding of the complex IV assembly factor Rcf2 and Mra1 interaction with an Atp9-translating mitoribosome induce Mra1 degradation, allowing synchronized maturation of complex IV and the ATP synthase. Failure of the stop-and-go mechanism results in cell death. MiRA controls OXPHOS assembly, ensuring correct stoichiometry of protein machineries encoded by two different genomes.

The thylakoid proton antiporter KEA3 regulates photosynthesis in response to the chloroplast energy status.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Protein interactome of 3',5'-cAMP reveals its role in regulating the actin cytoskeleton.

Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3',5'-cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3',5'-cAMP, we used a chemo-proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3',5'-cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3',5'-cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3',5'-cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3',5'-cAMP supplementation affected actin organization by inducing actin-bundling. Consistent with these results, the increase in 3',5'-cAMP levels, obtained either by feeding or by chemical modulation of 3',5'-cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3',5'-cAMP, as demonstrated using a positional isomer 2',3'-cAMP, and true for the nanomolar 3',5'-cAMP concentrations reported for plant cells. In vitro characterization of the 3',5'-cAMP-actin pairing argues against a direct interaction between actin and 3',5'-cAMP. Alternative mechanisms by which 3',5'-cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3',5'-cAMP interactome, as well as functional insight into 3',5'-cAMP-mediated regulation in plants.

Genomic instability in individuals with sex determination defects and germ cell cancer.

The ability to transmit genetic information through generations depends on the preservation of genome integrity. Genetic abnormalities affect cell differentiation, causing tissue specification defects and cancer. We addressed genomic instability in individuals with Differences of Sex Development (DSD), characterized by gonadal dysgenesis, infertility, high susceptibility for different types of cancer, especially Germ Cell Tumors (GCT), and in men with testicular GCTs. Whole proteome analysis of leukocytes, supported by specific gene expression assessment, and dysgenic gonads characterization, uncovered DNA damage phenotypes with altered innate immune response and autophagy. Further examination of DNA damage response revealed a reliance on deltaTP53, which was compromised by mutations in the transactivation domain in DSD-individuals with GCT. Accordingly, drug-induced rescue of DNA damage was achieved by autophagy inhibition but not by stabilization of TP53 in DSD-individuals' blood in vitro. This study elucidates possibilities for prophylactic treatments of DSD-individuals, as well as new diagnostic approaches of GCT.

Hello darkness, my old friend: 3-KETOACYL-COENZYME A SYNTHASE4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis thaliana.

Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.

3-Mercaptopyruvate sulfur transferase is a protein persulfidase.

Protein S-persulfidation (P-SSH) is recognized as a common posttranslational modification. It occurs under basal conditions and is often observed to be elevated under stress conditions. However, the mechanism(s) by which proteins are persulfidated inside cells have remained unclear. Here we report that 3-mercaptopyruvate sulfur transferase (MPST) engages in direct protein-to-protein transpersulfidation reactions beyond its previously known protein substrates thioredoxin and MOCS3/Uba4, associated with H2S generation and transfer RNA thiolation, respectively. We observe that depletion of MPST in human cells lowers overall intracellular protein persulfidation levels and identify a subset of proteins whose persulfidation depends on MPST. The predicted involvement of these proteins in the adaptation to stress responses supports the notion that MPST-dependent protein persulfidation promotes cytoprotective functions. The observation of MPST-independent protein persulfidation suggests that other protein persulfidases remain to be identified.

Don't let go: co-fractionation mass spectrometry for untargeted mapping of protein-metabolite interactomes.

The chemical complexity of metabolomes goes hand in hand with their functional diversity. Small molecules have many essential roles, many of which are executed by binding and modulating the function of a protein partner. The complex and dynamic protein-metabolite interaction (PMI) network underlies most if not all biological processes, but remains under-characterized. Herein, we highlight how co-fractionation mass spectrometry (CF-MS), a well-established approach to map protein assemblies, can be used for proteome and metabolome identification of the PMIs. We will review recent CF-MS studies, discuss the main advantages and limitations, summarize the available CF-MS guidelines, and outline future challenges and opportunities.

Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes Using a Single-Step Affinity Purification.

Cellular protein-metabolite interactions (PMI), for decades relatively overlooked, are seeing a golden age in recent years. To facilitate simultaneous characterization of PMI and protein-protein interactions (PPI) of a given protein ("bait"), we developed a protocol that utilizes antibody-assisted affinity purification (AP) followed by liquid chromatography-mass spectrometry (LC-MS). Aside from its speed, simplicity, and adaptability to a variety of biological systems, its main strength lies in the parallel identification, in a near-physiological environment, of a given protein's protein and small-molecule partners.

PROMIS: Co-fractionation Mass Spectrometry for Analysis of Protein-Metabolite Interactions.

The roles of small molecules in every aspect of life have been gaining increased recognition. Many are known to exert their effect by binding proteins-but a comprehensive overview of protein-metabolite interactions (PMIs) is missing. Recently we devised a non-targeted method for detecting PMIs using size-exclusion chromatography followed by proteomic and metabolomic analysis: PROMIS. Under test this method was able to identify known PMIs such as enzyme-cofactor complexes as well as novel ones.

Rewiring of the protein-protein-metabolite interactome during the diauxic shift in yeast.

In budding yeast Saccharomyces cerevisiae, the switch from aerobic fermentation to respiratory growth is separated by a period of growth arrest, known as the diauxic shift, accompanied by a significant metabolic rewiring, including the derepression of gluconeogenesis and the establishment of mitochondrial respiration. Previous studies reported hundreds of proteins and tens of metabolites accumulating differentially across the diauxic shift transition. To assess the differences in the protein-protein (PPIs) and protein-metabolite interactions (PMIs) yeast samples harvested in the glucose-utilizing, fermentative phase, ethanol-utilizing and early stationary respiratory phases were analysed using isothermal shift assay (iTSA) and a co-fractionation mass spectrometry approach, PROMIS. Whereas iTSA monitors changes in protein stability and is informative towards protein interaction status, PROMIS uses co-elution to delineate putative PPIs and PMIs. The resulting dataset comprises 1627 proteins and 247 metabolites, hundreds of proteins and tens of metabolites characterized by differential thermal stability and/or fractionation profile, constituting a novel resource to be mined for the regulatory PPIs and PMIs. The examples discussed here include (i) dissociation of the core and regulatory particle of the proteasome in the early stationary phase, (ii) the differential binding of a co-factor pyridoxal phosphate to the enzymes of amino acid metabolism and (iii) the putative, phase-specific interactions between proline-containing dipeptides and enzymes of central carbon metabolism.

PROMISed: A novel web-based tool to facilitate analysis and visualization of the molecular interaction networks from co-fractionation mass spectrometry (CF-MS) experiments.

Co-fractionation mass spectrometry (CF-MS)-based approaches enable cell-wide identification of protein-protein and protein-metabolite complexes present in the cellular lysate. CF-MS combines biochemical separation of molecular complexes with an untargeted mass-spectrometry-based proteomics and/or metabolomics analysis of the obtained fractions, and is used to delineate putative interactors. CF-MS data are a treasure trove for biological discovery. To facilitate analysis and visualization of original or publically available CF-MS datasets, we designed PROMISed, a user-friendly tool available online via https://myshiny.mpimp-golm.mpg.de/PDP1/ or as a repository via https://github.com/DennisSchlossarek/PROMISed. Specifically, starting with raw fractionation profiles, PROMISed (i) contains activities for data pre-processing and normalization, (ii) deconvolutes complex fractionation profiles into single, distinct peaks, (iii) identifies co-eluting protein-protein or protein-metabolite pairs using user-defined correlation methods, and (iv) performs co-fractionation network analysis. Given multiple CF-MS datasets, for instance representing different environmental condition, PROMISed allows to select for proteins and metabolites that differ in their elution profile, which may indicate change in the interaction status. But it also enables the identification of protein-protein and protein-metabolite pairs that co-elute together across multiple datasets. PROMISed enables users to (i) easily adjust parameters at each step of the analysis, (ii) download partial and final results, and (iii) select among different data-visualization options. PROMISed renders CF-MS data accessible to a broad scientific audience, allowing users with no computational or statistical background to look for novel protein-protein and protein-metabolite complexes for further experimental validation.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

Functional characterization of proton antiport regulation in the thylakoid membrane.

During photosynthesis, energy is transiently stored as an electrochemical proton gradient across the thylakoid membrane. The resulting proton motive force (pmf) is composed of a membrane potential (ΔΨ) and a proton concentration gradient (ΔpH) and powers the synthesis of ATP. Light energy availability for photosynthesis can change very rapidly and frequently in nature. Thylakoid ion transport proteins buffer the effects that light fluctuations have on photosynthesis by adjusting pmf and its composition. Ion channel activities dissipate ΔΨ, thereby reducing charge recombinations within photosystem II. The dissipation of ΔΨ allows for increased accumulation of protons in the thylakoid lumen, generating the signal that activates feedback downregulation of photosynthesis. Proton export from the lumen via the thylakoid K+ exchange antiporter 3 (KEA3), instead, decreases the ΔpH fraction of the pmf and thereby reduces the regulatory feedback signal. Here, we reveal that the Arabidopsis (Arabidopsis thaliana) KEA3 protein homo-dimerizes via its C-terminal domain. This C-terminus has a regulatory function, which responds to light intensity transients. Plants carrying a C-terminus-less KEA3 variant show reduced feed-back downregulation of photosynthesis and suffer from increased photosystem damage under long-term high light stress. However, during photosynthetic induction in high light, KEA3 deregulation leads to an increase in carbon fixation rates. Together, the data reveal a trade-off between long-term photoprotection and a short-term boost in carbon fixation rates, which is under the control of the KEA3 C-terminus.

Global mapping of protein-metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity.

Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/ . By interpolating PROMIS with the list of predicted protein-metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.

Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana.

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant (raptor1b), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b, can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes.

PROMIS: Global Analysis of PROtein-Metabolite Interactions.

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein-small molecule interactions in biological systems remains unknown. PROtein-metabolite interactions using size separation (PROMIS) is a method for studying protein-small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography-mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule-protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis.

Emerging strategies for the identification of protein-metabolite interactions.

Interactions between biological molecules enable life. The significance of a cell-wide understanding of molecular complexes is thus obvious. In comparison to protein-protein interactions, protein-metabolite interactions remain under-studied. However, this has been gradually changing due to technological progress. Here, we focus on the interactions between ligands and receptors, the triggers of signalling events. While the number of small molecules with proven or proposed signalling roles is rapidly growing, most of their protein receptors remain unknown. Conversely, there are numerous signalling proteins with predicted ligand-binding domains for which the identities of the metabolite counterparts remain elusive. Here, we discuss the current biochemical strategies for identifying protein-metabolite interactions and how they can be used to characterize known metabolite regulators and identify novel ones.