Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

Abstract

Dominant missense mutations in the human serine protease FAM111A underlie perinatally lethal gracile bone dysplasia and Kenny-Caffey syndrome 1-3 , yet how FAM111A mutations lead to disease is not known. We show that FAM111A proteolytic activity suppresses DNA replication and transcription by displacing key effectors of these processes from chromatin, triggering rapid programmed cell death by Caspase-dependent apoptosis to potently undermine cell viability. Patient-associated point mutations in FAM111A exacerbate these phenotypes by hyperactivating its intrinsic protease activity. Moreover, FAM111A forms a complex with the uncharacterized homologous serine protease FAM111B, point mutations in which cause a hereditary fibrosing poikiloderma syndrome 4 , and we demonstrate that disease-associated FAM111B mutants display amplified proteolytic activity and phenocopy the cellular impact of deregulated FAM111A catalytic activity. Thus, patient-associated FAM111A and FAM111B mutations may drive multisystem disorders via a common gain-offunction mechanism that relieves inhibitory constraints on their protease activities to powerfully undermine cellular fitness. Main textDominant missense mutations in human FAM111A underlie Kenny-Caffey syndrome and gracile bone dysplasia, which present with a broad spectrum of severe growth abnormalities with variable expressivity, including thin and brittle bones, dwarfism, facial dysmorphism and splenic hypoplasia 1-3 . FAM111A is a prospective host restriction factor that has been linked to DNA replication 5-7 but whose precise cellular function remains poorly understood. In addition to a PCNA-binding PIP box, FAM111A contains a functionally uncharacterized C-terminal serine protease domain, which

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“…The human FAM111A protein (NP001299838) is a 611 amino acid serine protease that contains a conserved catalytic triad [reviewed in Welter and Machida, 2022] with chymotrypsin-like specificity at the C-terminus and an autocleavage site between Phe334 and Gly335 (Hoffmann et al, 2020;Kojima et al, 2020). Its known functions include 1) interacting directly with PCNA and nascent chromatin to load PCNA at the DNA replication fork; 2) removing protein obstacles at the replication fork to prevent stalling and promote DNA synthesis; and 3) acting as a host restriction factor in antiviral defense (Kojima et al, 2020;Nie et al, 2021) against SV40 polyomavirus (Fine et al, 2012;Tarnita et al, 2018), orthopoxvirus (Panda et al, 2017), and Zika virus (Ren et al, 2021).…”

“…Its known functions include 1) interacting directly with PCNA and nascent chromatin to load PCNA at the DNA replication fork; 2) removing protein obstacles at the replication fork to prevent stalling and promote DNA synthesis; and 3) acting as a host restriction factor in antiviral defense (Kojima et al, 2020;Nie et al, 2021) against SV40 polyomavirus (Fine et al, 2012;Tarnita et al, 2018), orthopoxvirus (Panda et al, 2017), and Zika virus (Ren et al, 2021). Depletion of FAM111A in virus-infected cells disrupts host cell nuclear pore proteins and barrier function, leading to increased formation of viral replication centers (Hoffmann et al, 2020;Nie et al, 2021;Welter and Machida, 2022).…”

“…In humans, most of the heterozygous de novo missense variants that have been identified result in 'gain of function' and hyperactivity of the protease (Hoffmann et al, 2020). Mutations in human FAM111A are associated with Kenny-Caffey Syndrome, type 2 (KCS2, OMIM #127000), in which patients exhibit increased bone density, growth retardation, macrocephaly, facial dysmorphism, hypoparathyroidism, and electrolyte imbalances, including hypocalcemia and hypomagnesemia (Isojima et al, 2014;Abraham et al, 2017) or Gracile Bone Dysplasia/ Osteocraniostenosis (GCLEB/OCS, OMIM #602361), characterized by skeletal/craniofacial malformations, and perinatal lethality (Unger et al, 2013;Muller et al, 2021;Rosato et al, 2022).…”

Loss of FAM111B protease mutated in hereditary fibrosing poikiloderma negatively regulates telomere length

“…FAM111A localizes to DNA replication forks via a PIP-box mediated interaction with PCNA, where it processes DNA-protein crosslinks in response to replication stress (Kojima et al, 2020). Like FAM111A, the FAM111B protease has a conserved catalytic triad (residues H490, D544, S650) and shows protease activity in vitro (Hoffmann et al, 2020). Even though FAM111B does not contain a PIP box sequence, similarly to FAM111A it interacts with PCNA and the RFC complex (Hoffmann et al, 2020).…”

“…Like FAM111A, the FAM111B protease has a conserved catalytic triad (residues H490, D544, S650) and shows protease activity in vitro (Hoffmann et al, 2020). Even though FAM111B does not contain a PIP box sequence, similarly to FAM111A it interacts with PCNA and the RFC complex (Hoffmann et al, 2020). FAM111B also possesses a centrally located FAM111B-specfic domain (112aa) of unknown function.…”