Functions and evolution of FAM111 serine proteases

Abstract

Proteolysis plays fundamental and regulatory roles in diverse cellular processes. The serine protease FAM111A (FAM111 trypsin-like peptidase A) emerged recently as a protease involved in two seemingly distinct processes: DNA replication and antiviral defense. FAM111A localizes to nascent DNA and plays a role at the DNA replication fork. At the fork, FAM111A is hypothesized to promote DNA replication at DNA-protein crosslinks (DPCs) and protein obstacles. On the other hand, FAM111A has also been identified as a host restriction factor for mutants of SV40 and orthopoxviruses. FAM111A also has a paralog, FAM111B, a serine protease with unknown cellular functions. Furthermore, heterozygous missense mutations in FAM111A and FAM111B cause distinct genetic disorders. In this review, we discuss possible models that could explain how FAM111A can function as a protease in both DNA replication and antiviral defense. We also review the consequences of FAM111A and FAM111B mutations and explore possible mechanisms underlying the diseases. Additionally, we propose a possible explanation for what drove the evolution of FAM111 proteins and discuss why some species have two FAM111 proteases. Altogether, studies of FAM111 proteases in DNA repair, antiviral defense, and genetic diseases will help us elucidate their functions and the regulatory mechanisms.

Keywords: DNA-protein crosslink (DPC); FAM111A; FAM111B; Kenny-Caffey Syndrome (KCS); POIKTMP; gracile bone dysplasia; protease; viral replication.

FIGURE 1

FAM111A and FAM111B protein domain structures and mutations in genetic disorders. (A) Schematic representations of FAM111A and FAM111B protein domains. Amino acid mutations in patients for KCS2 (top) and GCLEB (bottom) in FAM111A and POIKTMP in FAM111B are notated. The two regions where patient mutations cluster, the hinge (cluster 1) and enzyme domain (cluster 2), are indicated. Catalytic triads are depicted in red. ^‡Compound heterozygous mutations. PIP: PCNA-interacting peptide box; UBL-1: Ubiquitin-like domain 1; UBL-2: Ubiquitin-like domain 2; Trypsin 2: Trypsin-like peptidase domain (Pfam ID: PF13365). (B) AlphaFold structural prediction of hinge region and enzyme domain of FAM111A (left) and FAM111B (right). Residues mutated in KCS2 (purple), GCLEB (blue) and POIKTMP (green) are indicated. Active site residues are colored in red and circled, and the Trypsin 2 domain is colored in orange. Structures are displayed using PyMOL (http://www.pymol.org/pymol).

FIGURE 2

FAM111A’s functions in cells and the implications of FAM111 mutations in human disorders. (A) Model of FAM111A’s roles in cells as an antiviral protease and DNA replication fork protein. (Left) FAM111A is a host restriction factor for host range mutants of SV40 and orthopoxviruses. Viral proteins LT (SV40) and SPI-1 (orthopoxvirus) are proposed to inhibit FAM111A protease activity and perturb its antiviral role. (Right) FAM111A functions at the DNA replication fork through its interaction with PCNA. PCNA: proliferating cell nuclear antigen. Figures were created with BioRender.com. (B) Possible mechanisms by which heterozygous mutations in FAM111A (KCS2/GCLEB) and FAM111B (POIKTMP) cause genetic disorders. (Left) Patient associated hyperactive mutants may degrade essential proteins (Model 1) or wild-type FAM111 proteins (Model 2). (Right) Patient associated inactive mutants (S541Y and S541P) of FAM111A may interfere with functions of wild-type enzymes by sequestering substrates. Figures were created with BioRender.com.

FIGURE 3

FAM111A ortholog sequence similarity network with FAM111B paralogs. The sequence similarity network (SSN) was generated using the Enzyme Function Initiative’s Enzyme Similarity Tool (https://efi.igb.illinois.edu/efi-est), using H. sapiens FAM111A protein as the query sequence and displayed with Cytoscape (https://cytoscape.org). All nodes from initial results are displayed, and only edges with 47–100% sequence identity between nodes are shown. Nodes are annotated by biological class, and nodes containing putative FAM111B paralogs are notated.

FIGURE 4

Phylogenetic inference of FAM111A orthologs. (A) A schematic representation of domain structures for representative orthologs of FAM111A. PIP: PCNA-interacting peptide box; UBL-1: Ubiquitin-like domain 1; UBL-2: Ubiquitin-like domain 2; Trypsin 2: Trypsin-like peptidase domain (Pfam ID: PF13365). (B) Bayesian phylogenetic prediction of FAM111A orthologs. A Bayesian phylogenetic tree inferred by MrBayes (http://nbisweden.github.io/MrBayes) was generated from a CLUSTAL Omega (https://www.ebi.ac.uk/Tools/msa/clustalo) FAM111A sequence alignment containing 14 representative species, selected from larger scale multiple sequence alignments and trees. Proteins included in analysis are Paramuricea clavata FAM111A (UniProt ID: A0A7D9LSX1), Branchiostoma belcheri FAM111A-like (UniProt ID: A0A6P4ZC58), Danio rerio FAM111A-like (UniProt ID: A0A8M2BD64), Salmo salar FAM111A (UniProt ID: A0A1S3N1P2), Leptobrachium leishanense Serine protease (A0A8C5WH67), Scyliorhinus torazame Serine protease (A0A401QCT3), Alligator sinensis FAM111A (A0A1U8DS27), Pantherophis guttatus FAM111A (A0A6P9C8G8), Sarcophilus harrisii FAM111A (G3VJT4), Loxodonta africana FAM111A (G3T8F5), Canis lupus familiaris FAM111A (A0A8C0SC36), Mus musculus FAM111A (Q9D2L9), Gorilla gorilla gorilla FAM111A (G3RXJ5), and Homo sapiens FAM111A (Q96PZ2). Among-site rate variation was set to inverse gamma and the outgroup was defined as Paramuricea clavata (determined as outgroup from prior tree analyses). Analysis ran until the average standard deviation of split frequencies approaches zero (<0.01). Node support values are Bayesian inference posterior probabilities, written as percentages. Species which contain FAM111B are highlighted in orange. The points where the PIP box and UBLs appear, as well as the occurrence of the FAM111 gene duplication are indicated. Scale bar indicates estimated substitutions per site. Figure was created with FigTree (http://tree.bio.ed.ac.uk/software/figtree) and BioRender.com.