Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization.

The prevailing model behind synapse development and specificity is that a multitude of adhesion molecules engage in transsynaptic interactions to induce pre- and postsynaptic assembly. How these extracellular interactions translate into intracellular signal transduction for synaptic assembly remains unclear. Here, we focus on a synapse organizing complex formed by immunoglobulin superfamily member 21 (IgSF21) and neurexin2α (Nrxn2α) that regulates GABAergic synapse development in the mouse brain. We reveal that the interaction between presynaptic Nrxn2α and postsynaptic IgSF21 is a high-affinity receptor-ligand interaction and identify a binding interface in the IgSF21-Nrxn2α complex. Despite being expressed in both dendritic and somatic regions, IgSF21 preferentially regulates dendritic GABAergic presynaptic differentiation whereas another canonical Nrxn ligand, neuroligin2 (Nlgn2), primarily regulates perisomatic presynaptic differentiation. To explore mechanisms that could underlie this compartment specificity, we targeted multiple signaling pathways pharmacologically while monitoring the synaptogenic activity of IgSF21 and Nlgn2. Interestingly, both IgSF21 and Nlgn2 require c-jun N-terminal kinase (JNK)-mediated signaling, whereas Nlgn2, but not IgSF21, additionally requires CaMKII and Src kinase activity. JNK inhibition diminished de novo presynaptic differentiation without affecting the maintenance of formed synapses. We further found that Nrxn2α knockout brains exhibit altered synaptic JNK activity in a sex-specific fashion, suggesting functional linkage between Nrxns and JNK. Thus, our study elucidates the structural and functional relationship of IgSF21 with Nrxn2α and distinct signaling pathways for IgSF21-Nrxn2α and Nlgn2-Nrxn synaptic organizing complexes in vitro. We therefore propose a revised hypothesis that Nrxns act as molecular hubs to specify synaptic properties not only through their multiple extracellular ligands but also through distinct intracellular signaling pathways of these ligands.

The 37TrillionCells initiative for improving global healthcare via cell-based interception and precision medicine: focus on neurodegenerative diseases.

One of the main burdens in the treatment of diseases is imputable to the delay between the appearance of molecular dysfunctions in the first affected disease cells and their presence in sufficient number for detection in specific tissues or organs. This delay obviously plays in favor of disease progression to an extent that makes efficient treatments difficult, as they arrive too late. The development of a novel medical strategy, termed cell-based interception and precision medicine, seeks to identify dysfunctional cells early, when tissue damages are not apparent and symptoms not yet present, and develop therapies to treat diseases early. Central to this strategy is the use of single-cell technologies that allow detection of molecular changes in cells at the time of phenotypical bifurcation from health to disease. In this article we describe a general procedure to support such an approach applied to neurodegenerative disorders. This procedure combines four components directed towards highly complementary objectives: 1) a high-performance single-cell proteomics (SCP) method (Detect), 2) the development of disease experimental cell models and predictive computational models of cell trajectories (Understand), 3) the discovery of specific targets and personalized therapies (Cure), and 4) the creation of a community of collaborating laboratories to accelerate the development of this novel medical paradigm (Collaborate). A global initiative named 37TrillionCells (37TC) was launched to advance the development of cell-based interception and precision medicine.

ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans.

BACKGROUND: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II). RESULTS: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 knockout only dysregulates 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical in folate uptake and hence neural tube development, is downregulated in the knockout intestine. We also identify nine deleterious mutations in the ARMC5 gene in 511 patients with myelomeningocele, a severe form of spina bifida. These mutations impair the interaction between ARMC5 and Pol II and reduce Pol II ubiquitination. CONCLUSIONS: Mutations in ARMC5 increase the risk of NTDs in mice and humans. ARMC5 is part of an E3 controlling the degradation of all 12 subunits of Pol II under physiological conditions. The Pol II pool size might have effects on NTD pathogenesis, and some of the effects might be via the downregulation of FOLH1. Additional mechanistic work is needed to establish the causal effect of the findings on NTD pathogenesis.

Drugs targeting the particle for arrangement of quaternary structure (PAQosome) and protein complex assembly.

INTRODUCTION: The PAQosome is a 12-subunit complex that acts as a co-factor of the molecular chaperones HSP90 and HSP70. This co-chaperone has been shown to participate in assembly and maturation of several protein complexes, including nuclear RNA polymerases, RNA processing factors, the ribosome, PIKKs, and others. Subunits of the PAQosome, adaptors, and clients have been reported to be involved in various diseases, making them interesting targets for drug discovery. AREA COVERED: In this review, the authors cover the detailed mechanisms of PAQosome and chaperone function. Specifically, the authors summarize the status of the PAQosome and some related chaperones and co-chaperones as candidate targets for drug discovery. Indeed, a number of compounds are currently being tested for the development of treatments against diseases, such as cancers and neurodegenerative conditions. EXPERT OPINION: Searching for new drugs targeting the PAQosome requires a better understanding of PAQosome subunit interactions and the discovery of new interaction partners. Thus, PAQosome subunit crystallization is an important experiment to initiate virtual screening against new target and the development of in silico tools such as AlphaFold-multimer could accelerate the search for new interaction partner and determine more rapidly the interaction pocket needed for virtual drug screening.

Biallelic pathogenic variants in POLR3D alter tRNA transcription and cause a hypomyelinating leukodystrophy: A case report.

RNA polymerase III-related leukodystrophy (POLR3-related leukodystrophy) is a rare, genetically determined hypomyelinating disease arising from biallelic pathogenic variants in genes encoding subunits of RNA polymerase III (Pol III). Here, we describe the first reported case of POLR3-related leukodystrophy caused by biallelic pathogenic variants in POLR3D, encoding the RPC4 subunit of Pol III. The individual, a female, demonstrated delays in walking and expressive and receptive language as a child and later cognitively plateaued. Additional neurological features included cerebellar signs (e.g., dysarthria, ataxia, and intention tremor) and dysphagia, while non-neurological features included hypodontia, hypogonadotropic hypogonadism, and dysmorphic facial features. Her MRI was notable for diffuse hypomyelination with myelin preservation of early myelinating structures, characteristic of POLR3-related leukodystrophy. Exome sequencing revealed the biallelic variants in POLR3D, a missense variant (c.541C > T, p.P181S) and an intronic splice site variant (c.656-6G > A, p.?). Functional studies of the patient's fibroblasts demonstrated significantly decreased RNA-level expression of POLR3D, along with reduced expression of other Pol III subunit genes. Notably, Pol III transcription was also shown to be aberrant, with a significant decrease in 7SK RNA and several distinct tRNA genes analyzed. Affinity purification coupled to mass spectrometry of the POLR3D p.P181S variant showed normal assembly of Pol III subunits yet altered interaction of Pol III with the PAQosome chaperone complex, indicating the missense variant is likely to alter complex maturation. This work identifies biallelic pathogenic variants in POLR3D as a novel genetic cause of POLR3-related leukodystrophy, expanding the molecular spectrum associated with this disease, and proposes altered tRNA homeostasis as a factor in the underlying biology of this hypomyelinating disorder.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

Riluzole partially restores RNA polymerase III complex assembly in cells expressing the leukodystrophy-causative variant POLR3B R103H.

The mechanism of assembly of RNA polymerase III (Pol III), the 17-subunit enzyme that synthesizes tRNAs, 5 S rRNA, and other small-nuclear (sn) RNAs in eukaryotes, is not clearly understood. The recent discovery of the HSP90 co-chaperone PAQosome (Particle for Arrangement of Quaternary structure) revealed a function for this machinery in the biogenesis of nuclear RNA polymerases. However, the connection between Pol III subunits and the PAQosome during the assembly process remains unexplored. Here, we report the development of a mass spectrometry-based assay that allows the characterization of Pol III assembly. This assay was used to dissect the stages of Pol III assembly, to start defining the function of the PAQosome in this process, to dissect the assembly defects driven by the leukodystrophy-causative R103H substitution in POLR3B, and to discover that riluzole, an FDA-approved drug for alleviation of ALS symptoms, partly corrects these assembly defects. Together, these results shed new light on the mechanism and regulation of human nuclear Pol III biogenesis.

ARMC5 is part of an RPB1-specific ubiquitin ligase implicated in adrenal hyperplasia.

ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA polymerase II (Pol II) as potential ARMC5-interacting proteins. Here, we show that ARMC5, CUL3 and RBX1 form an active E3 ligase complex specific for RPB1. ARMC5, CUL3, and RBX1 formed an active E3 specific for RPB1. Armc5 deletion caused a significant reduction in RPB1 ubiquitination and an increase in an accumulation of RPB1, and hence an enlarged Pol II pool in normal tissues and organs. The compromised RPB1 degradation did not cause generalized Pol II stalling nor depressed transcription in the adrenal glands but did result in dysregulation of a subset of genes, with most upregulated. We found RPB1 to be highly expressed in the adrenal nodules from patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) harboring germline ARMC5 mutations. Mutant ARMC5 had altered binding with RPB1. In summary, we discovered that wildtype ARMC5 was part of a novel RPB1-specific E3. ARMC5 mutations resulted in an enlarged Pol II pool, which dysregulated a subset of effector genes. Such an enlarged Pol II pool and gene dysregulation was correlated to adrenal hyperplasia in humans and KO mice.

Recurrent chromosomal translocations in sarcomas create a megacomplex that mislocalizes NuA4/TIP60 to Polycomb target loci.

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.

Whole Exome/Genome Sequencing Joint Analysis of a Family with Oligogenic Familial Hypercholesterolemia.

Autosomal Dominant Hypercholesterolemia (ADH) is a genetic disorder caused by pathogenic variants in LDLR, APOB, PCSK9 and APOE genes. We sought to identify new candidate genes responsible for the ADH phenotype in patients without pathogenic variants in the known ADH-causing genes by focusing on a French family with affected and non-affected members who presented a high ADH polygenic risk score (wPRS). Linkage analysis, whole exome and whole genome sequencing resulted in the identification of variants p.(Pro398Ala) in CYP7A1, p.(Val1382Phe) in LRP6 and p.(Ser202His) in LDLRAP1. A total of 6 other variants were identified in 6 of 160 unrelated ADH probands: p.(Ala13Val) and p.(Aps347Asn) in CYP7A1; p.(Tyr972Cys), p.(Thr1479Ile) and p.(Ser1612Phe) in LRP6; and p.(Ser202LeufsTer19) in LDLRAP1. All six probands presented a moderate wPRS. Serum analyses of carriers of the p.(Pro398Ala) variant in CYP7A1 showed no differences in the synthesis of bile acids compared to the serums of non-carriers. Functional studies of the four LRP6 mutants in HEK293T cells resulted in contradictory results excluding a major effect of each variant alone. Within the family, none of the heterozygous for only the LDLRAP1 p.(Ser202His) variant presented ADH. Altogether, each variant individually does not result in elevated LDL-C; however, the oligogenic combination of two or three variants reveals the ADH phenotype.

Unphosphorylated Form of the PAQosome Core Subunit RPAP3 Binds Ribosomal Preassembly Complexes to Modulate Ribosome Biogenesis.

The PAQosome (particle for arrangement of quaternary structure) is a 12-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely, the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using the expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119, or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances the binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, the silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modification is a mechanism of client selection.

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein.

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein-protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

Discovery of Antivirals Using Phage Display.

The latest coronavirus disease outbreak, COVID-19, has brought attention to viral infections which have posed serious health threats to humankind throughout history. The rapid global spread of COVID-19 is attributed to the increased human mobility of today's world, yet the threat of viral infections to global public health is expected to increase continuously in part due to increasing human-animal interface. Development of antiviral agents is crucial to combat both existing and novel viral infections. Recently, there is a growing interest in peptide/protein-based drug molecules. Antibodies are becoming especially predominant in the drug market. Indeed, in a remarkably short period, four antibody therapeutics were authorized for emergency use in COVID-19 treatment in the US, Russia, and India as of November 2020. Phage display has been one of the most widely used screening methods for peptide/antibody drug discovery. Several phage display-derived biologics are already in the market, and the expiration of intellectual property rights of phage-display antibody discovery platforms suggests an increment in antibody drugs in the near future. This review summarizes the most common phage display libraries used in antiviral discovery, highlights the approaches employed to enhance the antiviral potency of selected peptides/antibody fragments, and finally provides a discussion about the present status of the developed antivirals in clinic.

Analysis of the SARS-CoV-2-host protein interaction network reveals new biology and drug candidates: focus on the spike surface glycoprotein and RNA polymerase.

Introduction: The COVID-19 pandemic originated from the emergence of anovel coronavirus, SARS-CoV-2, which has been intensively studied since its discovery in order to generate the knowledge necessary to accelerate the development of vaccines and antivirals. Of note, many researchers believe there is great potential in systematically identifying host interactors of viral factors already targeted by existing drugs.Areas Covered: Herein, the authors discuss in detail the only available large-scale systematic study of the SARS-CoV-2-host protein-protein interaction network. More specifically, the authors review the literature on two key SARS-CoV-2 drug targets, the Spike surface glycoprotein, and the RNA polymerase. The authors also provide the reader with their expert opinion and future perspectives.Expert opinion: Interactions made by viral proteins with host factors reveal key functions that are likely usurped by the virus and, as aconsequence, points to known drugs that can be repurposed to fight viral infection and collateral damages that can exacerbate various disease conditions in COVID-19.

POLR3-related leukodystrophy: How do mutations affecting RNA polymerase III subunits cause hypomyelination?

Hypomyelinating leukodystrophies are a group of genetic disorders characterized by insufficient myelin deposition during development. A subset of hypomyelinating leukodystrophies, named RNA polymerase III (Pol III or POLR3)-related leukodystrophy or 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) leukodystrophy, was found to be caused by biallelic variants in genes encoding subunits of the enzyme Pol III, including POLR3A, POLR3B, POLR3K, and POLR1C. Pol III is one of the three nuclear RNA polymerases that synthesizes small non-coding RNAs, such as tRNAs, 5S RNA, and others, that are involved in the regulation of essential cellular processes, including transcription, translation and RNA maturation. Affinity purification coupled with mass spectrometry (AP-MS) revealed that a number of mutations causing POLR3-related leukodystrophy impair normal assembly or biogenesis of Pol III, often causing a retention of the unassembled subunits in the cytoplasm. Even though these proteomic studies have helped to understand the molecular defects associated with leukodystrophy, how these mutations cause hypomyelination has yet to be defined. In this review we propose two main hypotheses to explain how mutations affecting Pol III subunits can cause hypomyelination.

De novo variants in POLR3B cause ataxia, spasticity, and demyelinating neuropathy.

POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.

Variants in LSM7 impair LSM complexes assembly, neurodevelopment in zebrafish and may be associated with an ultra-rare neurological disease.

Leukodystrophies, genetic neurodevelopmental and/or neurodegenerative disorders of cerebral white matter, result from impaired myelin homeostasis and metabolism. Numerous genes have been implicated in these heterogeneous disorders; however, many individuals remain without a molecular diagnosis. Using whole-exome sequencing, biallelic variants in LSM7 were uncovered in two unrelated individuals, one with a leukodystrophy and the other who died in utero. LSM7 is part of the two principle LSM protein complexes in eukaryotes, namely LSM1-7 and LSM2-8. Here, we investigate the molecular and functional outcomes of these LSM7 biallelic variants in vitro and in vivo. Affinity purification-mass spectrometry of the LSM7 variants showed defects in the assembly of both LSM complexes. Lsm7 knockdown in zebrafish led to central nervous system defects, including impaired oligodendrocyte development and motor behavior. Our findings demonstrate that variants in LSM7 cause misassembly of the LSM complexes, impair neurodevelopment of the zebrafish, and may be implicated in human disease. The identification of more affected individuals is needed before the molecular mechanisms of mRNA decay and splicing regulation are added to the categories of biological dysfunctions implicated in leukodystrophies, neurodevelopmental and/or neurodegenerative diseases.

Upstream ORF-Encoded ASDURF Is a Novel Prefoldin-like Subunit of the PAQosome.

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.

Ser-Phosphorylation of PCSK9 (Proprotein Convertase Subtilisin-Kexin 9) by Fam20C (Family With Sequence Similarity 20, Member C) Kinase Enhances Its Ability to Degrade the LDLR (Low-Density Lipoprotein Receptor).

OBJECTIVE: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. CONCLUSIONS: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.