Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62.

Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

Cholinesterase Inhibitory Arisugacins L-Q from a Penicillium sp. Isolate Obtained through a Citizen Science Initiative and Their Activities in a Phenotype-Based Zebrafish Assay.

Phenotype-based screening of a fungal extract library yielded an active sample from a Penicillium sp. isolate that impaired zebrafish motility. Bioassay-guided purification led to the identification of 14 meroterpenoids including six new metabolites, arisugacins L-Q (4, 5, 8, and 12-14), seven known arisugacins (1-3, 6, 7, 9, and 10), and one known terreulactone (11). Their structures were determined using a combination of NMR and HRESIMS data, evidence secured from theoretical and experimental ECD spectra, and the modified Mosher's method. The purified compounds were tested in zebrafish embryos, as well as in vitro for cholinesterase inhibition activities. Compound 12 produced defects in myotome structure (metameric muscle, which is critical for locomotion) in vivo and showed the most potent and selective acetylcholinesterase inhibitory activity with an IC50 of 191 nM in vitro. The phenotype assay was also used to reveal bioactivities for several previously reported arisugacins, which had failed to show activity in prior cell-based and in vitro testing. This study demonstrates that utilization of the zebrafish phenotype assay is an effective approach for the identification of bioactive extracts, is compatible with the bioassay-guided compound purification strategies, and offers a valuable tool for probing complex natural product sources to detect bioactive small molecules with potential therapeutic or other commercial applications.

Functional characterization of CNOT3 variants identified in familial adenomatous polyposis adenomas.

Germline mutations in the tumor suppressor Adenomatous Polyposis Coli (APC) define Familial Adenomatous Polyposis (FAP), the genetic predisposition to developing adenomatous polyps. Recent sequencing of FAP adenomas have challenged established dogma that APC mutations alone represent the adenoma mutational landscape because recurrent somatic mutations in non-WNT pathway genes were also discovered. In particular, one of these novel genes, CNOT3, presented E20K and E70K mutations that are predicted to be deleterious in silico. We utilized zebrafish embryos to determine if these mutations affect CNOT3 function and perform novel biology in an APC-dependent pathway in vivo. Human CNOT3 (hCNOT3) and E20K mRNA injection rescued zebrafish cnot3a knockdown lordosis phenotype while E70K did not. In the FAP apcmcr zebrafish model, we show that ctbp1, but not retinoic acid, regulates cnot3a expression. Injection of hCNOT3 and E20K, but not E70K, to homozygous apcmcr zebrafish initiated gut differentiation while cnot3a knockdown in wildtype embryos led to decreased intestinal development and differentiation. Finally, targeted sequencing of 37 additional FAP adenomas revealed CNOT3 mutations in 20% of these samples. Overall, our work supports a mechanism where CTBP1 regulates CNOT3 and that overall CNOT3 perturbation could work in concert with germline APC mutations in advancing adenomas to a more transformed state prior to progression to adenocarcinoma.

A metabolic switch controls intestinal differentiation downstream of Adenomatous polyposis coli (APC).

Elucidating signaling pathways that regulate cellular metabolism is essential for a better understanding of normal development and tumorigenesis. Recent studies have shown that mitochondrial pyruvate carrier 1 (MPC1), a crucial player in pyruvate metabolism, is downregulated in colon adenocarcinomas. Utilizing zebrafish to examine the genetic relationship between MPC1 and Adenomatous polyposis coli (APC), a key tumor suppressor in colorectal cancer, we found that apc controls the levels of mpc1 and that knock down of mpc1 recapitulates phenotypes of impaired apc function including failed intestinal differentiation. Exogenous human MPC1 RNA rescued failed intestinal differentiation in zebrafish models of apc deficiency. Our data demonstrate a novel role for apc in pyruvate metabolism and that pyruvate metabolism dictates intestinal cell fate and differentiation decisions downstream of apc.

Juxtaposition of chemical and mutation-induced developmental defects in zebrafish reveal a copper-chelating activity for kalihinol F.

A major hurdle in using complex systems for drug screening is the difficulty of defining the mechanistic targets of small molecules. The zebrafish provides an excellent model system for juxtaposing developmental phenotypes with mechanism discovery using organism genetics. We carried out a phenotype-based screen of uncharacterized small molecules in zebrafish that produced a variety of chemically induced phenotypes with potential genetic parallels. Specifically, kalihinol F caused an undulated notochord, defects in pigment formation, hematopoiesis, and neural development. These phenotypes were strikingly similar to the zebrafish mutant, calamity, an established model of copper deficiency. Further studies into the mechanism of action of kalihinol F revealed a copper-chelating activity. Our data support this mechanism of action for kalihinol F and the utility of zebrafish as an effective system for identifying therapeutic and target pathways.

A novel conus peptide ligand for K+ channels.

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.