Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of ( S )-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249 -encoded transmembrane (TM) domain–containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics and transcriptomics data to infer the evolutionary history of DNases and investigate their biological significance. Both DNase1 and DNase2 families have expanded in vertebrates since ~ 650 million years ago before the divergence of jawless and jawed vertebrates. DNase1, DNase1L1, and DNase1L3 co-existed in jawless fish, whereas DNase1L2 originated in amniotes by tandem duplication of DNase1. Among the non-human DNases, DNase1L4 and newly identified DNase1L5 derived from early duplications that were lost in terrestrial vertebrates. The ancestral gene of the DNase2 family, DNase2b, has been conserved in synteny with the Uox gene across 700 million years of animal evolution,while DNase2 originated in jawless fish. DNase1L1 acquired a GPI-anchor for plasma membrane attachment in bony fishes, and DNase1L3 acquired a C-terminal basic peptide for the degradation of microparticle DNA in jawed vertebrates. The appearance of DNase1L2, with a distinct low pH optimum and skin localization, is among the amniote adaptations to life on land. The expansion of the DNase repertoire in vertebrates meets the diversified demand for DNA debris removal in complex multicellular organisms.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H₂O₂-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with β-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

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