The SNX8 is a sorting nexin protein involved in intracellular molecular traffic from the early endosomes to the trans-golgi network. It is suggested that it acts as an adaptor protein in events related to immune response and cholesterol regulation, for example. As a protein of the SNXs family, the SNX8 is formed of 465 aminoacids and presents a BAR domain and a PX domain which are very relevant in relation to its functions. Furthermore, SNX8 study is motivated by its medical significance in relation to diseases such as Alzheimer's Disease, cancer, neurodevelopmental malformations and to its role in fighting against viral infections.
Structure Sorting nexins (SNXs)
SNX8 belongs to the sorting nexin family of proteins, which mainly contain two functional membrane-binding that allow SNXs to have different roles in endosomal sorting and protein trafficking thanks to its membrane curvature ability. To begin with, SNX-PX is a distinct phosphoinositide (PI)-binding domain. The preferential interaction of this domain with membrane lipids makes the main function of SNX-PX the targeting of proteins to phosphatidylinositol-3-phosphate (PI(3)P) to endosomes. On the other hand, the BAR (Bin/amphiphysin/Rvs) domain is a key regulator of phosphoinositide-mediated, tubular-based endosomal sorting. Accordingly, this domain also dimerizes to sense, stabilize and induce membrane curvature. The SNX-BAR proteins that contain both domains are a part of phosphoinositide-enriched, high-curvature tubular micro-domains of the endo-lysosomal network.
The mammalian genome contains 12 genes coding for SNX-BAR proteins (SNX1, SNX2, SNX4, SNX9, SNX18, SNX32 and SNX33). Other domains, such as PDZ (postsynaptic density protein-95, discs-large, zona occludens-1), SH3 (Src homology 3) and RA (Ras-associated), are involved in protein-protein interactions.
SNX8
The SNX8 protein, even though is very similar to the other sorting nexins, presents a domain structure which resembles the most to SNX1's and SNX9's; for this reason, although its terciary structure remains unknown, it theoretically resembles that of SNX9 shown in the model above. Overall, the SNX8 protein is integrated by one unique peptide chain that has 465 amino acids with a molecular mass of 52.569 Da.
PX Domain-containing N-terminus
SNX8 contains a PX domain in its N-terminus, which is located between amino acids 71 and 181. A homology domain with yeast's PX domain is localized between amino acids 75 and 178 within this same domain. As it is a phosphoinositide-binding domain, it is important to highlight amino acids 109, 135 and 148 as residues directly related to phosphatidylinositol 3-phosphate since being specific binding sites, constituting a phosphoinositid binding site with a span of 40 amino acids. Furthermore, it includes a pair of phosphorylable tyrosines in positions 95 and 126 that are key in its function in the IFNγ-triggered IKKβ-mediated noncanonical signaling pathway. Overall, the PX domain main function is to target SNX8 mainly to early endosomes and other membranes rich in phosphatidylinositol 3-phosphate phospholipids.
BAR Domain-containing C-terminus
SNX contains a BAR domain in its C-terminus, which is located between amino acids 256 and 440. Its ability to form coatings in membranes in order to induce their curvature is key in SNX8 participation in tubular structures formation. Furthermore, SNX8 C-terminus contains a threonine in position 452 and a serine in position 456 which can go through post-traductional changes that induces its phosphorylation, resulting in a phosphothreonine and a phosphoserine. Therefore, there are classified as phosphorylation sites.
MVP1 ortholog SNX8 has a yeast ortholog protein, the MVP1 encoded by the also homolog gene Mvp1p, which also plays a role by mediating transport of cargo to the vacuolar and lysosomal compartments. For this reason, its investigation can lead to a better understanding of SNX8 functions in human cells.
Biological functions and its molecular mechanisms SNX8 is thought to be an adaptor protein involved in the endosome-to-Golgi transport pathway, participating in endocytosis and endosomal sorting and signaling. It downregulates retrograde transport of intracellular proteins from the early endosome compartment to the trans-Golgi network in a retromer-mediated manner. SNX8 is therefore localized in early endosomes, as its colocalization with components of the retromer such as SNX1, SNX2, Vps26 and Vps35 has been demonstrated by some studies (and also with EEA1). Furthermore, the dynamics of endosomal structures with SNX8-enriched membrane domains are regulated by the opposite motor proteins dynein-1 containing LIC1 and kinesin-1, both of which allow SNX8-mediated cargo movement through the cytosol by exerting forces on these structures. The biological functions of SNX8 that have been studied, all of which involve its role in intracellular endosomal transport, are explained in more detail in the following sections.
Innate immune response Cytosolic RNA virus-triggered response SNX8 is a key component in the induction of downstream effector antiviral gene transcription in response to RNA viruses, as it is necessary for RLR-mediated activation of the IFNβ promoter that leads to host defense in a dose-dependent manner. It positively regulates the aggregation and activation of VISA, a key adaptor protein involved in the innate immune response against this type of infections; this process is critical in the recruitment of other signaling components. Although SNX8's standard localization includes the cytoplasm and other compartments such as the ER, the ER-Golgi intermediate compartment, the Golgi apparatus or endosomes, it is partially localized at mitochondria. Furthermore, RNA viral infections cause the translocation of SNX8 from the cytosol to the mitochondria. During the early stage of the infection, SNX8 association with VISA increases above constitutive levels (degradation of VISA in later stages results in a reduction of this phenomenon). Structurally, the C-terminus transmembrane domain of VISA and both N-terminus PX domain and C-terminus of SNX8 are necessary for this linkage. The formation of the SNX8-VISA complex potentiates VISA prion-like polymerization and aggregation. After VISA activation, its accumulation allows the recruitment of signaling components which play a role in RLR-mediated antiviral response activation such as the intermediates of this pathway TBK1, IRF3, IκBα or ISRE. IRF3 cooperative activation with NF-κB transcription factor through phosphorylation leads to the induction of the IFNβ promoter transcription. Examples of genes whose RNA virus-triggered expression is stimulated by SNX8 are IFNB1, ISG56 and IL6 (being IL6 and IFNB1 related to cytokine secretion).
In addition, SNX8 also plays a role in RIG-I containing CARD domain-mediated and MDA5-mediated activation of the IFNβ promoter, since VISA works as an intermediate for both signaling pathways. The mechanism for SNX8 recruitment to VISA remains unclear, although two options have been suggested: translocation of viral RNA-bound RIG-I or MDA5 to VISA may result in a conformational switch that would increase its affinity for SNX8 or the RNA virus may induce post-translational modifications of one of these proteins allowing the translocation of SNX8 to mitochondria for its interaction with VISA.
Cytosolic DNA virus-triggered response
SNX8 is a key component in the induction of downstream effector antiviral gene transcription in response to DNA viruses, as it is necessary for MITA-mediated activation of the IFNβ promoter in a dose-dependent manner. It regulates the activation of MITA, a central adaptor protein in the innate immune response to DNA viral infections that activates and onsets the antiviral response, by allowing its association with the class III phosphatylinositol 3 kinase VPS34-containing translocon machinery to form the MITA-Vps34 translocation complex; this linkage is crucial for MITA transport from the ER to perinuclear microsomal punctuate structures, which are induced by infection of viruses such as HSV-1, via Golgi mediated intracellular traffic. This MITA transport pathway is vital for the immune response to start. SNX8 is suggested to play a role in MITA's activation through this transport pathway, modulating its phosphorylation at serine in position 366 and recruiting the transcription factor IRF3 whose activation is important in the activation of the IFNβ promoter transcription. This hypothesis is supported by the fact that MITA, SNX8 and VPS34 colocalize in the cytoplasm, and that SNX8 localization in ER, ER-Golgi intermediate compartment, Golgi and endosomes is similar to that of MITA. Examples of genes whose DNA virus-triggered expression is stimulated by SNX8 are IFNB1, ISG56, CXCL10 and IL6 (being IFNB1 and IL6 related to cytokine secretion).
IFNγ-triggered IKKβ-mediated noncanonical signaling pathway SNX8 is a component of an IKKβ-mediated noncanonical signaling pathway triggered by the interferon gamma, which takes places parallelly to the more known IFNγ-induced JAK-STAT1 mediated pathway. Overall, it participates in a series of chemical reactions and molecular interactions that lead to the selective promotion of a particular subset of downstream effector gene transcription that encode IFNγ-induced GTPases of the GBP family, which participate in host defense against intracellular pathogens, and secretion proteins of the chemokine family such as CXCL9, CXCL10, and CXCL11 which present direct antimicrobial activity. In addition, SNX8 enhances the IFNγ-induced activation of the IRF1 promoter in a dose-dependent manner. Specifically, the steps of this pathway that are regulated by SNX8 are the following. The reception of IFNγ activates Janus kinase 1, resulting in the stimulation of its association with Sxn8 above standard constitutive levels. The link between these two protein within the JAK1-SNX8 complex allows JAK1 to catalyse SNX8's tyrosines phosphorylation in positions 95 and 126. This phosphorylation activates the JAK1-SNX, and the SNX8 acts as an adaptor or scaffolding protein by permitting the recruitment of the inhibitor of nuclear factor-kappa-B kinase subunit beta (IKKβ) to JAK1 in the JAK1-SNX8-IKKβ complex. This linkage is essential for IKKβ activation through autophosphorylation at serine in position 177 (as SNX8 lacks enzymatic activity) and further dimerization and oligomerization.
Cholesterol regulation SNX8 protein regulates cholesterol levels as an activator of the SREBPs (Sterol Regulatory Element Binding Proteins), which is a family of transcription factors that control the expression of enzymes needed for the synthesis and uptake of fatty acids, endogenous cholesterol, triacylglycerides and phospholipids; this results in an overall regulation of intracellular lipid homeostasis. Although its precise mechanism of action remains unknown, data suggests that SNX8 produces changes in cholesterol distribution through regulation of the SREBP transcriptional activity by modulating intracellular traffic events rather than by interacting with proteins of the SREBP pathway like INSIG or SREBP cleavage-activating protein (SCAP). For example, it is unclear if SNX8 has a direct participation in the transport of SREBP pathway components or if it regulates endosomal and lysosomal compartmentalization through the production of cholesterol cargoes. This last possibility is supported by the fact that the ability of altering membrane curvature is shared by some proteins of the SNXs family.