Fig 2: K-48 deubiquitinating activity of USP48 wt and pMet415Ile mutant. (A) Deubiquitination assay with immunopurified FLAG-tagged USP48 variants and purified K48-linked ubiquitin chains, containing 2 to 7 ubiquitin moieties. Upper panel shows the immunoblot of the deubiquitination reactions using anti-ubiquitin antibodies. Lower panel shows purified USP48 proteins. (B) Deubiquitination assay and purification of His6-ubiquitinated histone H2A extracted from HeLa cells, transiently overexpressing FLAG-tagged USP48 variants and HA-tagged histone H2A. (C) Deubiquitination reaction using total extracts of cells transiently overexpressed HA-tagged Gli1 and FLAG-tagged USP48 variants in HeLa cells showing enhanced deubiquitination of Gli1 by mutant USP48 resulting in an increase in Gli1 levels. - = naive cells, WT = cells transfected with USP48 WT, Mut = cells transfected with USP48 Met415Ile mutant, CA: cells transfected with the Cys98Ala catalytically inactive USP48 variant.

Fig 3: Theoretical 3D model of the catalytic subunit of USP48 protein variants. Structure of the USP domain of USP48 for the wt (A and zoomed-in view in B) and the p.Met415Ile variant (C and zoomed-in view in D), developed using the SWISS-MODEL server based application (based on Uniprot sequence: Q86UV5) showing in violet Met415 and Ile415, respectively, and in orange the nearest neighboring amino acids, Ala182 and Phe103. The distance between the amino acids is represented by dotted lines and for Met415 amounts to 3.16 Å to Ala182 and 2.02 Å to Phe103, while it is increased for the mutated Ile415 to 7.24 Å to Ala182 and 4.20 Å to Phe103. The catalytic triad Cys98/His353/Asn370 is represented in blue in the foreground.

Fig 4: Hypothesized mechanisms leading to corticotroph tumor formation involving the different recurrently mutated genes discovered in CD. The 3 genes most frequently mutated in CD (USP8, USP48, and TP53) are shown in the context of (patho)physiological corticotroph cell regulation. CRH acts on Nur and AP1 binding elements on the POMC promoter to activate transcription downstream to the cAMP/extracellular signal-regulated kinase (ERK) signaling pathways. Activated epidermal growth factor receptor acts via ERK1/2 to stimulate POMC transcription; activating USP8 mutations potentiate this effect by deubiquitinating and rescuing the receptor from lysosomal degradation.13 SHH binding to Patched (Ptch1) relieves suppression of Smoothened (Smo) allowing for Gli1 activation; SHH and CRH crosstalk at Gli1 level to stimulate POMC transcription and ACTH secretion.19 We hypothesize that activating mutations in the USP48 deubiquitinase lead to increased levels of Gli1, which enhances basal and CRH-induced POMC transcription via an unknown at present mechanism. In Drosophila S2 cells, USP8 was shown to prevent Smo ubiquitination.32 Activating USP48 mutations may also lead to increased deubiquitination of histone H2A and thus to a decreased recruitment of DNA repair factors in case of DNA damage and increased tumorigenesis potential. This effect can also be triggered by inactivating mutations in TP53.40 Green lines represent physiological, red lines pathological activity, and gray lines mechanism of action shown in other cell systems but not yet proven in corticotroph tumor cells. Dotted lines present indirect effects.