A Single Kinase Generates the Majority of the Secreted Phosphoproteome.

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.

Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis.

The family with sequence similarity 20, member C (Fam20C) has recently been identified as the Golgi casein kinase. Fam20C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam20C is potentially an upstream regulator of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), because humans with FAM20C mutations and Fam20C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam20C regulates FGF23 is unknown. Here we show that Fam20C directly phosphorylates FGF23 on Ser(180), within the FGF23 R(176)XXR(179)/S(180)AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated.

Identification of a mammalian-type phosphatidylglycerophosphate phosphatase in the Eubacterium Rhodopirellula baltica.

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.

Structural and functional analysis of PTPMT1, a phosphatase required for cardiolipin synthesis.

PTPMT1 (PTP localized to the Mitochondrion 1) is a member of the protein tyrosine phosphatase superfamily that is localized exclusively to the mitochondrion. We recently reported that PTPMT1 dephosphorylates phosphatidylglycerol phosphate, an essential intermediate of cardiolipin biosynthesis. To gain further insights into the molecular basis of PTPMT1 function, we determined the crystal structures of the phosphatase domain of PTPMT1. PTPMT1 exhibits a canonical protein tyrosine phosphatase domain fold, resembling many dual-specificity phosphatases such as phosphatase and tensin homolog and vaccinia H1-related phosphatase. We also determined the structure of the catalytically inactive phosphatase in complex with a surrogate substrate, phosphatidylinositol 5-phosphate, which sheds light on the substrate recognition and specificity of PTPMT1. Comparison of the apo and substrate-bound structures of PTPMT1 suggests that it undergoes significant conformational change during catalysis, and we further demonstrated that an evolutionarily conserved EEYE loop is important for its activity.

UBLCP1 is a 26S proteasome phosphatase that regulates nuclear proteasome activity.

Protein degradation by the 26S proteasome is a fundamental process involved in a broad range of cellular activities, yet how proteasome activity is regulated remains poorly understood. We report here that ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1) is a 26S proteasome phosphatase that regulates nuclear proteasome activity. UBLCP1 directly interacts with the proteasome via its UBL domain and is exclusively localized in the nucleus. UBLCP1 dephosphorylates the 26S proteasome and inhibits proteasome activity in vitro. Knockdown of UBLCP1 in cells promotes 26S proteasome assembly and selectively enhances nuclear proteasome activity. Our results describe the first identified proteasome-specific phosphatase and uncover a unique mechanism for phosphoregulation of the proteasome.

Single Cell Multi-Omics of an iPSC Model of Human Sinoatrial Node Development Reveals Genetic Determinants of Heart Rate and Arrhythmia Susceptibility.

Cellular heterogeneity within the sinoatrial node (SAN) is functionally important but has been difficult to model in vitro , presenting a major obstacle to studies of heart rate regulation and arrhythmias. Here we describe a scalable method to derive sinoatrial node pacemaker cardiomyocytes (PCs) from human induced pluripotent stem cells that recapitulates differentiation into distinct PC subtypes, including SAN Head, SAN Tail, transitional zone cells, and sinus venosus myocardium. Single cell (sc) RNA-sequencing, sc-ATAC-sequencing, and trajectory analyses were used to define epigenetic and transcriptomic signatures of each cell type, and to identify novel transcriptional pathways important for PC subtype differentiation. Integration of our multi-omics datasets with genome wide association studies uncovered cell type-specific regulatory elements that associated with heart rate regulation and susceptibility to atrial fibrillation. Taken together, these datasets validate a novel, robust, and realistic in vitro platform that will enable deeper mechanistic exploration of human cardiac automaticity and arrhythmia.

Isolation and characterization of human embryonic stem cell-derived heart field-specific cardiomyocytes unravels new insights into their transcriptional and electrophysiological profiles.

AIMS: We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. METHODS AND RESULTS: Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. CONCLUSIONS: We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.

Direct Cardiac Reprogramming: Progress and Promise.

The human adult heart lacks a robust endogenous repair mechanism to fully restore cardiac function after insult; thus, the ability to regenerate and repair the injured myocardium remains a top priority in treating heart failure. The ability to efficiently generate a large number of functioning cardiomyocytes capable of functional integration within the injured heart has been difficult. However, the ability to directly convert fibroblasts into cardiomyocyte-like cells both in vitro and in vivo offers great promise in overcoming this problem. In this review, we describe the insights and progress that have been gained from the investigation of direct cardiac reprogramming. We focus on the use of key transcription factors and cardiogenic genes as well as on the use of other biological molecules such as small molecules, cytokines, noncoding RNAs, and epigenetic modifiers to improve the efficiency of cardiac reprogramming. Finally, we discuss the development of safer reprogramming approaches for future clinical application.

Analysis of cardiomyocyte clonal expansion during mouse heart development and injury.

The cellular mechanisms driving cardiac tissue formation remain poorly understood, largely due to the structural and functional complexity of the heart. It is unclear whether newly generated myocytes originate from cardiac stem/progenitor cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Here, we identify the source of new cardiomyocytes during mouse development and after injury. Our findings suggest that cardiac progenitors maintain proliferative potential and are the main source of cardiomyocytes during development; however, the onset of αMHC expression leads to reduced cycling capacity. Single-cell RNA sequencing reveals a proliferative, "progenitor-like" population abundant in early embryonic stages that decreases to minimal levels postnatally. Furthermore, cardiac injury by ligation of the left anterior descending artery was found to activate cardiomyocyte proliferation in neonatal but not adult mice. Our data suggest that clonal dominance of differentiating progenitors mediates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late embryonic development and shortly after birth.

Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.

Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis.

PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.

A family of bacterial cysteine protease type III effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes.

Bacterial phytopathogens employ a type III secretion system to deliver effector proteins into the plant cell to suppress defense pathways; however, the molecular mechanisms and subcellular localization strategies that drive effector function largely remain a mystery. Here, we demonstrate that the plant plasma membrane is the primary site for subcellular localization of the Pseudomonas syringae effector AvrPphB and five additional cysteine protease family members. AvrPphB and two AvrPphB-like effectors, ORF4 and NopT, autoproteolytically process following delivery into the plant cell to expose embedded sites for fatty acylation. Host-dependent lipidation of these three effectors directs plasma membrane localization and is required for the avirulence activity of AvrPphB. Surprisingly, the AvrPphB-like effectors RipT, HopC1, and HopN1 utilize an acylation-independent mechanism to localize to the cellular plasma membrane. Although some AvrPphB-like effectors employ acylation-independent localization strategies, others hijack the eukaryotic lipidation machinery to ensure plasma membrane localization, illustrating the diverse tactics employed by type III effectors to target specific subcellular compartments.