LIG_VCP_SHPBox_1

The Endoplasmic Reticulum (ER) is an important eukaryotic cell organelle that has various functions, including the synthesis of proteins for export and quality control of nascent proteins. Newly synthesized proteins undergo folding and post-translational modifications in the ER. However, some proteins may not reach their native folded state. The ERAD (ER-Associated Degradation) mechanism acts as a protein quality control and removes these misfolded proteins. ERAD enables ER processing to distinguish the properly and improperly folded proteins in the ER lumen and then extracts them through membrane channels (dislocation or retrotranslocation) in an energy-dependent manner for delivery to cytosolic proteasomes. Nearly all ERAD substrates are ubiquitinated prior to their degradation and these ubiquitin chains provide a binding site for VCP (Valosin-Containing Protein). Thus ERAD is essential for ER homeostasis and correct functioning by degrading misfolded proteins (Hwang,2018).
Vertebrate VCP (also known as p97 or TERA for Transitional endoplasmic reticulum ATPase; Ter94 in fly, CDC48 in yeast) is a hexameric multidomain protein belonging to the functionally highly diverse AAA+ (ATPases Associated with diverse cellular Activities) superfamily of proteins. This large group of proteins drive numerous cell biological processes by converting chemical energy into mechanical energy (Khan,2022). As noted in PAXdb, VCP is a highly expressed protein, routinely observed to be amongst the top 5% of cellular proteins. VCP is likely an essential protein in all eukaryotes (Muller,2007). It is reported to be involved in a plethora of intracellular processes with the help of various co-factor proteins that specifically recruit ubiquitylated substrates. A tight control of VCP cofactor specificity and diversity as well as the assembly of higher-order VCP-cofactor complexes is accomplished by various regulatory mechanisms, which include bipartite binding, binding site competition, changes in oligomeric assemblies, and nucleotide-induced conformational changes (Hanzelmann,2017). More than 40 co-factor proteins have been identified so far, and most of them are multidomain proteins composed of specific VCP binding modules and additional domains that have functions in the recognition of ubiquitylated target proteins or possess catalytic domains or transmembrane domains (Buchberger,2015). Based on their functions, cofactors can be divided into three major classes: (i) Substrate-recruiting co-factors, such as the UFD1/NPL4 complex, link substrates to VCP and contain VCP interacting motifs and an additional ubiquitin binding domain that target ubiquitylated substrates; (ii) Substrate processing cofactors like ubiquitin (E3) ligases, deubiquitinases (DUBs) and cytosolic peptide N-glycanases (PNGase) process ubiquitylated, and N-glycosylated substrates; (iii) Regulatory cofactors like UBXD4, ASPL and SVIP sequester or recycle VCP hexamers. A few cofactors bind via their PUB or PUL domain to the unstructured C-terminal tail of VCP while the majority of the cofactors interact with the N-terminal VCP domain (CDC48_N; PF02359), often termed P97N, either via a UBX/UBXL globular domain or any one of three linear motifs, called VCP-Interacting Motif (VIM), VCP-Binding Motif (VBM), and SHP Box (named after yeast protein Shp1) (Hanzelmann,2017). In the nucleus, VCP is recruited for DNA damage repair by the SHP box protein Spartan (SPRTN) which specifically cleaves DNA-protein cross-links (Kroning,2022).

VIM and VBM are arginine-rich motifs found in several VCP cofactors with diverse functions (Buchberger,2015). The VCP CDC48_N domain has two subdomains or “lobes”. The interdomain cleft between the Nn and Nc lobes of CDC48_N provides a sterically unopposed interface for the interaction of the various VCP cofactor proteins. Despite the absence of significant sequence similarity, the VBM and VIM motifs bind partially overlapping sites at the interdomain cleft of the N domain. Hence, one N domain can only interact with one of these motifs at a time, reducing the complexity of cofactor interactions to a combinatorial problem of six N domains per VCP hexamer. The SHP box motif interacts with the C-terminal Nc/NTD subdomain of VCP CDC48_N, a site distinct from that to which the other VCP ligands bind (Lim,2016). Competition for N domain binding has been experimentally verified for various combinations of cofactors possessing different binding modules, e.g. SHP/UBX-VIM (p47-UBXD1; Kern,2009), VIM-VBM (SVIP-HRD1; Liu,2013), VIM–SHP/UBXL (gp78 – UFD1‐NPL4; Ballar,2006). Among them, SVIP is the only cofactor that binds with high affinity to all six N domains through the VIM motif forming the 6:6 stoichiometry. It is an efficient competitor for N domain cofactors and acts as a negative regulator of the ERAD pathway.

ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in more than 60 diseases including neurological dysfunction, cancer and cystic fibrosis (Guerriero,2012). Mutations in VCP are also causative of three protein aggregation diseases, Multisystem Proteinopathy (MSP), Familial Amyotrophic Lateral Sclerosis (FALS) and Charcot-Marie-Tooth Disease Type2Y (CMT2Y) (Ye,2017).

Many viruses exploit ERAD processes to promote their viral replication and to avoid detection by the immune response. The herpesviruses manipulate the immune response by the degradation of Major Histocompatibility complex (MHC-1) through retrotranslocation by the viral proteins US2 and US11. Likewise, the accessory protein Vpu of HIV induces CD4 degradation through the ERAD process helping to promote HIV infection. Many bacterial toxins also use the ERAD to invade host cells, e.g., the cholera Toxin protein employs the ERAD to enter the cytosol (Morito,2015). Different virus strains of the Nidovirales order, including the coronaviruses, use the ER-derived tuning vesicles (EDEMosomes) and double-membrane vesicles (DMVs) to sequester their double-stranded RNA from cytosolic sensors that will trigger interferon production and innate immunity (Zhang,2020; Noack,2014). These observations suggest that there might be the potential for bacterial and viral proteins to harbour VCP interacting motifs to interfere with ERAD processes.