Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Loss of Sec-1 Family Domain-Containing 1 (scfd1) Causes Severe Cardiac Defects and Endoplasmic Reticulum Stress in Zebrafish.

Dilated cardiomyopathy (DCM) is a common heart muscle disorder that frequently leads to heart failure, arrhythmias, and death. While DCM is often heritable, disease-causing mutations are identified in only ~30% of cases. In a forward genetic mutagenesis screen, we identified a novel zebrafish mutant, heart and head (hahvcc43), characterized by early-onset cardiomyopathy and craniofacial defects. Linkage analysis and next-generation sequencing identified a nonsense variant in the highly conserved scfd1 gene, also known as sly1, that encodes sec1 family domain-containing 1. Sec1/Munc18 proteins, such as Scfd1, are involved in membrane fusion regulating endoplasmic reticulum (ER)/Golgi transport. CRISPR/Cas9-engineered scfd1vcc44 null mutants showed severe cardiac and craniofacial defects and embryonic lethality that recapitulated the phenotype of hahvcc43 mutants. Electron micrographs of scfd1-depleted cardiomyocytes showed reduced myofibril width and sarcomere density, as well as reticular network disorganization and fragmentation of Golgi stacks. Furthermore, quantitative PCR analysis showed upregulation of ER stress response and apoptosis markers. Both heterozygous hahvcc43 mutants and scfd1vcc44 mutants survived to adulthood, showing chamber dilation and reduced ventricular contraction. Collectively, our data implicate scfd1 loss-of-function as the genetic defect at the hahvcc43 locus and provide new insights into the role of scfd1 in cardiac development and function.

Identifying novel genes for amyotrophic lateral sclerosis by integrating human brain proteomes with genome-wide association data.

BACKGROUND: Genome-Wide Association Studies (GWAS) have identified numerous risk genes for Amyotrophic Lateral Sclerosis (ALS); however, the mechanisms by which these loci confer ALS risk are uncertain. This study aims to identify novel causal proteins in the brains of patients with ALS using an integrative analytical pipeline. METHODS: Using the datasets of Protein Quantitative Trait Loci (pQTL) (NpQTL1 = 376, NpQTL2 = 152), expression QTL (eQTL) (N = 452), and the largest ALS GWAS (NALS=27,205, NControls = 110,881), we performed a systematic analytical pipeline including Proteome-Wide Association Study (PWAS), Mendelian Randomization (MR), Bayesian colocalization, and Transcriptome-Wide Association Study (TWAS) to identify novel causal proteins for ALS in the brain. RESULTS: Using PWAS, we found that the altered protein abundance of 12 genes in the brain was associated with ALS. Three genes (SCFD1, SARM1 and CAMLG) were identified as lead causal genes for ALS with solid evidence (False discovery rate < 0.05, in MR analysis; PPH4 > 80% for Bayesian colocalization). Specifically, an increased abundance of SCFD1 and CAMLG led to an increased risk of ALS, whereas a higher abundance of SARM1 led to a decreased risk of developing ALS. TWAS showed that SCFD1 and CAMLG were related to ALS at the transcriptional level. CONCLUSIONS: SCFD1, CAMLG, and SARM1 exhibited robust associations and causality with ALS. The study findings provide novel clues for identifying potential therapeutic targets in ALS. Further studies are required to explore the mechanisms underlying the identified genes.

Loss of amyotrophic lateral sclerosis risk factor SCFD1 causes motor dysfunction in Drosophila.

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease mostly resulting from a complex interplay between genetic, environmental and lifestyle factors. Common genetic variants in the Sec1 Family Domain Containing 1 (SCFD1) gene have been associated with increased ALS risk in the most extensive genome-wide association study (GWAS). SCFD1 was also identified as a top-most significant expression Quantitative Trait Locus (eQTL) for ALS. Whether loss of SCFD1 function directly contributes to motor system dysfunction remains unresolved. Here we show that moderate gene silencing of Slh, the Drosophila orthologue of SCFD1, is sufficient to cause climbing and flight defects in adult flies. A more severe knockdown induced a significant reduction in larval mobility and profound neuromuscular junction (NMJ) deficits prior to death before metamorphosis. RNA-seq revealed downregulation of genes encoding chaperones that mediate protein folding downstream of Slh ablation. Our findings support the notion that loss of SCFD1 function is a meaningful contributor to ALS and disease predisposition may result from erosion of the mechanisms protecting against misfolding and protein aggregation.

Acetylation of SCFD1 regulates SNARE complex formation and autophagosome-lysosome fusion.

SCFD1 (sec1 family domain containing 1) was recently shown to function in autophagosome-lysosome fusion, and multiple studies have demonstrated the regulatory impacts of acetylation (a post-translational modification) on macroautophagy/autophagy. Here, we demonstrate that both acetylation- and phosphorylation-dependent mechanisms control SCFD1's function in autophagosome-lysosome fusion. After detecting a decrease in the extent of SCFD1 acetylation under autophagy-stimulated conditions, we found that KAT2B/PCAF catalyzes the acetylation of residues K126 and K515 of SCFD1; we also showed that these two residues are deacetylated by SIRT4. Importantly, we showed that AMPK-controlled SCFD1 phosphorylation strongly disrupts the capacity of SCFD1 to interact with KAT2B, thus ensuring that the SCFD1 acetylation level remains low. Finally, we demonstrated that SCFD1 acetylation inhibits autophagic flux, specifically by blocking STX17-SNAP29-VAMP8 SNARE complex formation. Thus, our study reveals a mechanism through which phosphorylation and acetylation modifications of SCFD1 mediate SNARE complex formation to regulate autophagosome maturation.ACLY: ATP citrate lyase; CREB: cAMP responsive element binding protein; EBSS: nutrient-deprivation medium; EP300: E1A binding protein p300; KAT5/TIP60: lysine acetyltransferase 5; HOPS: homotypic fusion and protein sorting; MS: mass spectroscopy; SCFD1: sec1 family domain containing 1; SM: Sec1/Munc18; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; UVRAG: UV radiation resistance associated.

An imaging-based RNA interference screen for modulators of the Rab6-mediated Golgi-to-ER pathway in mammalian cells.

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

MTOR-mediates hepatic lipid metabolism through an autophagic SNARE complex.

The STX17-SNAP29-VAMP8 SNARE complex mediates autophagosome-lysosome fusion. Our recent study showed that MTOR directly phosphorylates VAMP8's T48 residue in nutrient-rich conditions. Phosphorylated VAMP8 inhibits autophagosome-lysosome fusion by blocking STX17-SNAP29-VAMP8 SNARE complex formation. Our study also showed that SCFD1 is a previously unrecognized macroautophagy/autophagy regulatory protein, which can be recruited by VAMP8 (in its non-phosphorylated form) to autolysosomes, where it promotes STX17-SNAP29-VAMP8 complex assembly - and consequently promotes autophagosome-lysosome fusion. Moreover, we observed that mice harboring a phosphomimic VAMP8 variant accumulate aberrantly high lipid levels in their livers. VAMP8 phosphorylation can disrupt autophagosome-lysosome fusion in the liver and thereby dysregulate lipid metabolism. Beyond providing insights into the molecular mechanisms of autophagosome maturation, our study suggests that modulating autophagic SNARE function may help treat liver lipid disorders.

Does SCFD1 rs10139154 Polymorphism Decrease Alzheimer's Disease Risk?

Α number of genetic variants have been associated with Alzheimer's disease (AD) susceptibility. Sec1 family domain-containing protein 1 (SCFD1) gene polymorphism rs10139154 has recently been implicated in the risk of developing amyotrophic lateral sclerosis (ALS). Similarities in the pathogenetic cascade of both diseases have also been described. The present study was designed to evaluate the possible contribution of SCFD1 rs10139154 to AD. A total of 327 patients with AD and an equal number of healthy controls were included in the study and genotyped for rs10139154. With logistic regression analyses, rs10139154 was examined for the association with the risk of developing AD. In the recessive mode, SCFD1 rs10139154 was associated with a decreased risk of developing AD (odds ratio (OR) (95% confidence interval (CI)) = 0.63 (0.40-0.97), p = 0.036). The current study provides preliminary evidence of the involvement of SCFD1 rs10139154 in the risk of developing AD.

An association study between SCFD1 rs10139154 variant and amyotrophic lateral sclerosis in a Chinese cohort.

BACKGROUND: A recent genome-wide association study (GWAS) demonstrated that the Sec1 family domain containing 1 (SCFD1) gene is associated with amyotrophic lateral sclerosis (ALS). The objective of our study was to investigate the association between the single nucleotide polymorphism (SNP) rs10139154 in the SCFD1 gene and ALS in a Chinese cohort. METHODS: A cohort of 1074 sporadic ALS (SALS) patients from the Department of Neurology at the West China Hospital of Sichuan University were genotyped for rs10139154 using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. In addition, 927 unrelated healthy controls (HCs) from the same region were included. RESULTS: After adjusting for age and sex, no significant differences in the genotype distributions and allele frequencies in the allelic, additive, dominant or recessive genetic models were found between SALS and HCs and between patients with spinal onset and bulbar onset. Remarkably, rs10139154 was shown to be associated with the age at onset (AAO) of ALS patients. Consistently, ALS patients with the "CC" genotype have an earlier mean AAO than that of patients with a "CG" and "CG + GG" genotype (p = 0.002 and 0.001, respectively). CONCLUSION: Our results suggest that there is a lack of association of SCFD1 rs10139154 with the risk for ALS in a large Chinese population, but this variant may modulate the age of onset of ALS. These findings add further evidence to the suspected implication of the SCFD1 gene in the pathogenesis of disease in our ALS population.

The Sec domain protein Scfd1 facilitates trafficking of ECM components during chondrogenesis.

Chondrogenesis in the developing skeleton requires transformation of chondrocytes from a simple mesenchymal condensation to cells with a highly enriched extracellular matrix (ECM). This transition is in part accomplished by alterations in the chondrocyte protein transport machinery to cope with both the increased amount and large size of ECM components. In a zebrafish mutagenesis screen to identify genes essential for cartilage development, we uncovered a mutant that disrupts the gene encoding Sec1 family domain containing 1 (scfd1). Homozygous scfd1 mutant embryos exhibit a profound craniofacial abnormality caused by a failure of chondrogenesis. Loss of scfd1 was found to hinder ER to Golgi transport of ECM proteins and is accompanied with activation of the unfolded protein response in chondrocytes. We further demonstrate a conserved role for Scfd1 in differentiation of mammalian chondrocytes, in which loss of either SCFD1 or STX18, a SLY1 interacting t-SNARE, severely impair transport of type II collagen. These results show that the existence of a specific export pathway, mediated by a complex containing SCFD1 and STX18 that plays an essential role in secretion of large ECM proteins during chondrogenesis.