No abstract

Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling.

MK-8666, a selective GPR40 agonist developed for the treatment of type 2 diabetes mellitus, was discontinued in phase I clinical trials due to liver safety concerns. To address whether chemically reactive metabolites played a causative role in the observed drug induced liver injury (DILI), we characterized the metabolism, covalent binding to proteins, and amino acid targets of MK-8666 in rat and human hepatocytes or cofactor-fortified liver microsomes. MK-8666 was primarily metabolized to an acyl glucuronide in hepatocytes of both species and a taurine conjugate in rat hepatocytes. Similar levels of covalent binding to proteins were observed in rat and human hepatocytes following incubation with [ 3 H]MK-8666. After protease digestion of hepatocyte pellets, amino acid adducts A1, A2, and A3 were identified as transacylated products with lysine, serine, and cysteine residues, respectively. Amino acid adducts A4a−c were identified as glycation adducts resulting from rearrangement of MK-8666−1-O-β-acyl glucuronide to ring-opened aldehydes which further condensed with lysine residues of proteins into imine adducts. Adducts A1−A3 and A4a−c were detected in rat and human liver microsomes fortified with UDPGA. Adducts A1−A3 were detected in rat and human liver microsomes fortified with CoA and ATP. Additionally, a trace amount of CoA thioester metabolite of MK-8666 and its transacylated GSH adduct were detected in human liver microsomes fortified with CoA, ATP, and GSH. Higher levels of covalent binding to protein were observed when [ 3 H]MK-8666 was incubated in liver microsomes supplemented with CoA and ATP compared to UDPGA. Addition of GSH attenuated levels of CoA thioester-mediated covalent binding by 41−45%. Collectively, these studies indicated that metabolism of the −COOH moiety of MK-8666 can form a reactive acyl glucuronide and an acyl CoA thioester, which covalently modifies proteins and may represent one causative mechanism of the observed DILI.

Pex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein’s trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p.

ABC-transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography, with regulation driven by the cytosolic domains, and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the protein defective in the most abundant rare inherited disease cystic fibrosis, by combining biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies a different mode of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.