Modeling protease-sensitive human pancreatic lipase mutations in the mouse ortholog.

Pancreatic lipase (PNLIP) is the major lipolytic enzyme secreted by the pancreas. A recent study identified human PNLIP variants P245A, I265R, F300L, S304F, and F314L in European chronic pancreatitis cohorts. Functional analyses indicated that the variants were normally secreted but exhibited reduced stability when exposed to pancreatic proteases. Proteolysis of the PNLIP variants yielded an intact C-terminal domain, while the N-terminal domain was degraded. The protease-sensitive PNLIP phenotype was strongly correlated with chronic pancreatitis, suggesting a novel pathological pathway underlying the disease. To facilitate preclinical mouse modeling, here we investigated how the human mutations affected the secretion and proteolytic stability of mouse PNLIP. We found that variants I265R, F300L, S304F, and F314L were secreted at high levels, while P245A had a secretion defect and accumulated inside the cells. Proteolysis experiments indicated that wild-type mouse PNLIP was resistant to cleavage, while variant I265R was readily degraded by mouse trypsin and chymotrypsin C. Variants F300L, S304F, and F314L were unaffected by trypsin but were slowly proteolyzed by chymotrypsin C. The proteases degraded the N-terminal domain of variant I265R, leaving the C-terminal domain intact. Structural analyses suggested that changes in stabilizing interactions around the I265R mutation site contribute to the increased proteolytic susceptibility of this variant. The results demonstrate that variant I265R is the best candidate for modeling the protease-sensitive PNLIP phenotype in mice.

Comprehensive functional characterization of complement factor I rare variant genotypes identified in the SCOPE geographic atrophy cohort.

Rare variants (RVs) in the gene encoding the regulatory enzyme complement factor I (CFI; FI) that reduce protein function or levels increase age-related macular degeneration risk. A total of 3357 subjects underwent screening in the SCOPE natural history study for geographic atrophy secondary to age-related macular degeneration, including CFI sequencing and serum FI measurement. Eleven CFI RV genotypes that were challenging to categorize as type I (low serum level) or type II (normal serum level, reduced enzymatic function) were characterized in the context of pure FI protein in C3b and C4b fluid phase cleavage assays and a novel bead-based functional assay (BBFA) of C3b cleavage. Four variants predicted or previously characterized as benign were analyzed by BBFA for comparison. In all, three variants (W51S, C67R, and I370T) resulted in low expression. Furthermore, four variants (P64L, R339Q, G527V, and P528T) were identified as being highly deleterious with IC50s for C3b breakdown >1 log increased versus the WT protein, while two variants (K476E and R474Q) were ∼1 log reduced in function. Meanwhile, six variants (P50A, T203I, K441R, E548Q, P553S, and S570T) had IC50s similar to WT. Odds ratios and BBFA IC50s were positively correlated (r = 0.76, p < 0.01), while odds ratios versus combined annotation dependent depletion (CADD) scores were not (r = 0.43, p = 0.16). Overall, 15 CFI RVs were functionally characterized which may aid future patient stratification for complement-targeted therapies. Pure protein in vitro analysis remains the gold standard for determining the functional consequence of CFI RVs.

Structural and functional analysis of two SHMT8 variants associated with soybean cyst nematode resistance.

Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5-phosphate-dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN-resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF-dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN-resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF-dependent catalytic activity relative to Essex SHMT8 (10- to 50-fold decrease in kcat /Km ) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF-substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants.

Congenital erythropoietic porphyria presenting with recurrent epistaxis: a case report.

BACKGROUND: Congenital erythropoietic porphyria (CEP), also known as pink tooth or Gunther disease, is a rare hereditary disorder caused by an enzyme mutation in the heme biosynthesis pathway, which leads to the accumulation of immature and non-physiological protoporphyrin rings in various tissues. CEP is characterized by sun-exposed bullous skin lesions, hemolytic anemia, red/brown urine, and teeth staining. CASE PRESENTATION: We present a unique case of a 10-year-old Asian boy with CEP who presented with recurrent epistaxis, an unusual presentation for this condition. Based on clinical presentation and laboratory findings, including elevated urine uroporphyrin and coproporphyrin I and III levels, microcytic anemia, a higher red cell distribution width (RDW), and a lower platelet count, a thorough assessment and detailed workup resulted in a diagnosis of CEP. The patient underwent a successful splenectomy and recovered without any complications. CONCLUSION: This case report aims to raise awareness among healthcare professionals about the uncommon and atypical presentation of CEP and its management options.

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic 'gatekeeper' enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Šresolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

Colorectal cancer-associated mutations impair EphB1 kinase function.

Erythropoietin-producing hepatoma (Eph) receptor tyrosine kinases regulate the migration and adhesion of cells that are required for many developmental processes and adult tissue homeostasis. In the intestinal epithelium, Eph signaling controls the positioning of cell types along the crypt-villus axis. Eph activity can suppress the progression of colorectal cancer (CRC). The most frequently mutated Eph receptor in metastatic CRC is EphB1. However, the functional effects of EphB1 mutations are mostly unknown. We expressed and purified the kinase domains of WT and five cancer-associated mutant EphB1 and developed assays to assess the functional effects of the mutations. Using purified proteins, we determined that CRC-associated mutations reduce the activity and stability of the folded structure of EphB1. By mammalian cell expression, we determined that CRC-associated mutant EphB1 receptors inhibit signal transducer and activator of transcription 3 and extracellular signal-regulated kinases 1 and 2 signaling. In contrast to the WT, the mutant EphB1 receptors are unable to suppress the migration of human CRC cells. The CRC-associated mutations also impair cell compartmentalization in an assay in which EphB1-expressing cells are cocultured with ligand (ephrin B1)-expressing cells. These results suggest that somatic mutations impair the kinase-dependent tumor suppressor function of EphB1 in CRC.

Neurodevelopmental disorder mutations in the purine biosynthetic enzyme IMPDH2 disrupt its allosteric regulation.

Inosine 5' monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report the identification of two additional missense variants in IMPDH2 from affected individuals and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation.

Mutation, food-grade expression, and characterization of a lactonase for zearalenone degradation.

Zearalenone (ZEN) is a mycotoxin that causes serious threats to human health. People are exposed to ZEN contamination externally and internally through many ways, while environmental-friendly strategies for efficient elimination of ZEN are urgently needed worldwide. Previous studies revealed that the lactonase Zhd101 from Clonostachys rosea can hydrolyze ZEN to low toxicity compounds. In this work, the enzyme Zhd101 was conducted with combinational mutations to enhance its application properties. The optimal mutant (V153H-V158F), named Zhd101.1, was selected and introduced into the food-grade recombinant yeast strain Kluyveromyces lactis GG799(pKLAC1-Zhd101.1), followed by induced expression and secretion into the supernatant. The enzymatic properties of this mutant were extensively examined, revealing a 1.1-fold increase in specific activity, as well as improved thermostability and pH stability, compared to the wild-type enzyme. The ZEN degradation tests and the reaction parameters optimization were carried out in both solutions and the ZEN-contaminated corns, using the fermentation supernatants of the food-grade yeast strain. Results showed that the degradation rates for ZEN by fermentation supernatants reached 96.9% under optimal reaction conditions and 74.6% in corn samples, respectively. These new results are a useful reference to zearalenone biodegradation technologies and indicated that the mutant enzyme Zhd101.1 has potential to be used in food and feed industries. KEY POINTS: • Mutated lactonase showed 1.1-fold activity, better pH stability than the wild type. • The strain K. lactis GG799(pKLAC1-Zhd101.1) and the mutant Zhd101.1 are food-grade. • ZEN degradation rates by supernatants reached 96.9% in solution and 74.6% in corns.

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism.

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases.

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

A single amino acid residue tunes the stability of the fully reduced flavin cofactor and photorepair activity in photolyases.

The UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 photoproducts), can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. The fully reduced flavin (hydroquinone, HQ) cofactor is required for the catalysis of both types of these photolyases. On the other hand, flavin cofactor in the semireduced state, semiquinone, can be utilized by photolyase homologs, the cryptochromes. However, the evolutionary process of the transition of the functional states of flavin cofactors in photolyases and cryptochromes remains mysterious. In this work, we investigated three representative photolyases (Escherichia coli CPD photolyase, Microcystis aeruginosa DASH, and Phaeodactylum tricornutum 6-4 photolyase). We show that the residue at a single site adjacent to the flavin cofactor (corresponding to Ala377 in E. coli CPD photolyase, hereafter referred to as site 377) can fine-tune the stability of the HQ cofactor. We found that, in the presence of a polar residue (such as Ser or Asn) at site 377, HQ was stabilized against oxidation. Furthermore, this polar residue enhanced the photorepair activity of these photolyases both in vitro and in vivo. In contrast, substitution of hydrophobic residues, such as Ile, at site 377 in these photolyases adversely affected the stability of HQ. We speculate that these differential residue preferences at site 377 in photolyase proteins might reflect an important evolutionary event that altered the stability of HQ on the timeline from expression of photolyases to that of cryptochromes.

Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA.

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.

Conventional protein kinase C in the brain: repurposing cancer drugs for neurodegenerative treatment?

Protein Kinase C (PKC) isozymes are tightly regulated kinases that transduce a myriad of signals from receptor-mediated hydrolysis of membrane phospholipids. They play an important role in brain physiology, and dysregulation of PKC activity is associated with neurodegeneration. Gain-of-function mutations in PKCα are associated with Alzheimer's disease (AD) and mutations in PKCγ cause spinocerebellar ataxia (SCA) type 14 (SCA14). This article presents an overview of the role of the conventional PKCα and PKCγ in neurodegeneration and proposes repurposing PKC inhibitors, which failed in clinical trials for cancer, for the treatment of neurodegenerative diseases.

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease.

ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA's substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to "tug on" and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.

Naturally occurring cancer-associated mutations disrupt oligomerization and activity of protein arginine methyltransferase 1 (PRMT1).

Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences.

Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis.

Xylanases produce xylooligosaccharides from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as the major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on the sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (kcat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures functional diversity in a limited number of protein variants.

From a dimer to a monomer: Construction of a chimeric monomeric isocitrate dehydrogenase.

Many isocitrate dehydrogenases (IDHs) are dimeric enzymes whose catalytic sites are located at the intersubunit interface, whereas monomeric IDHs form catalytic sites with single polypeptide chains. It was proposed that monomeric IDHs were evolved from dimeric ones by partial gene duplication and fusion, but the evolutionary process had not been reproduced in laboratory. To construct a chimeric monomeric IDH from homo-dimeric one, it is necessary to reconstitute an active center by a duplicated region; to properly link the duplicated region to the rest part; and to optimize the newly formed protein surface. In this study, a chimeric monomeric IDH was successfully constructed by using homo-dimeric Escherichia coli IDH as a start point by rational design and site-saturation mutagenesis. The ~67 kDa chimeric enzyme behaved as a monomer in solution, with a Km of 61 µM and a kcat of 15 s-1 for isocitrate in the presence of NADP+ and Mn2+ . Our result demonstrated that dimeric IDHs have a potential to evolve monomeric ones. The evolution of the IDH family was also discussed.

PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

APOBEC3-induced mutation of the hepatitis virus B DNA genome occurs during its viral RNA reverse transcription into (-)-DNA.

APOBEC3s are innate single-stranded DNA cytidine-to-uridine deaminases that catalyze mutations in both pathogen and human genomes with significant roles in human disease. However, how APOBEC3s mutate a single-stranded DNA that is available momentarily during DNA transcription or replication in vivo remains relatively unknown. In this study, utilizing hepatitis B virus (HBV) viral mutations, we evaluated the mutational characteristics of individual APOBEC3s with reference to the HBV replication process through HBV whole single-strand (-)-DNA genome mutation analyses. We found that APOBEC3s induced C-to-T mutations from the HBV reverse transcription start site continuing through the whole (-)-DNA transcript to the termination site with variable efficiency, in an order of A3B >> A3G > A3H-II or A3C. A3B had a 3-fold higher mutation efficiency than A3H-II or A3C with up to 65% of all HBV genomic cytidines being converted into uridines in a single mutation event, consistent with the A3B localized hypermutation signature in cancer, namely, kataegis. On the other hand, A3C expression led to a 3-fold higher number of mutation-positive HBV genome clones, although each individual clone had a lower number of C-to-T mutations. Like A3B, A3C preferred both 5'-TC and 5'-CC sequences, but to a lesser degree. The APOBEC3-induced HBV mutations were predominantly detected in the HBV rcDNA but were not detectable in other intermediates including HBV cccDNA and pgRNA by primer extension of their PCR amplification products. These data demonstrate that APOBEC3-induced HBV genome mutations occur predominantly when the HBV RNA genome was reversely transcribed into (-)-DNA in the viral capsid.

The ABCs of the atypical Fam20 secretory pathway kinases.

The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.