Current knowledge on the biology of squamous cell vulvar carcinoma (VSCC) is limited. We aimed to identify protein markers of VSCC tumors that would permit to stratify patients by progression risk. Early-stage tumors from patients who progressed (progVSCC) and from those who were disease-free (d-fVSCC) during follow-up, along with normal vulvar tissues were examined by mass spectrometry-based proteomics. Differentially expressed proteins (DEPs) were then verified in solid tissues and blood samples of patients with VSCC tumors and vulvar premalignant lesions. In progVSCC vs. d-fVSCC tumors, the immune response was the most over-represented Gene Ontology category for the identified DEPs. Pathway profiling suggested bacterial infections to be linked to aggressive VSCC phenotypes. High Mobility Group AT-Hook 2 (HMGA2) and Proteinase 3 (PRTN3) were revealed as proteins predicting VSCC progression. HMGA2 and PRTN3 abundances are associated with an aggressive phenotype, and hold promise as markers for VSCC patient stratification. It appears that vulvovaginal microflora disturbances trigger an inflammatory response contributing to cancer progression, suggesting that bacterial rather than viral infection status should be considered in the development of targeted therapies in VSCC.

The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen–deuterium exchange mass spectrometry to define Gorab’s interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab’s C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer’s coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6’s Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab’s dual role.

The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6in vitroandin vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.

The ability to relocalize proteins to defined subcellular locations presents a powerful tool to examine protein-protein interactions that can overcome a tendency of non-targeted exogenous proteins to form inaccessible aggregates. Here, we show that a 24-amino-acid sequence from the Drosophila proapoptotic protein Hid's tail anchor (HTA) domain can target exogenous proteins to the mitochondria in Drosophila cells. We use this HTA tag to target the Drosophila centriole cartwheel protein Sas6 to the mitochondria, and show that both exogenous and endogenous Gorab can be co-recruited from the Golgi to the new mitochondrial site. This accords with our previous observation that monomeric Drosophila Gorab binds Sas6 to become centriole associated with a 50-fold greater affinity than dimeric Gorab binds Rab6 to become localized at the Golgi. Strikingly, Drosophila Sas6 can bind both Drosophila Gorab and its human GORAB ortholog, whereas human SAS6 is unable to bind either GORAB or Gorab. We discuss these findings in relation to the evolutionary conservation of Gorab and the divergence of Sas6, possibly reflecting known differences in persistence of the cartwheel in the centriole duplication cycle of fly and human cells.