Peroxisome biogenesis and molecular defects in peroxisome assembly disorders

Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively. Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family. We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD. We also cloned PEX1 by screening of human liver cDNA library, using ZP107. PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same a CG-I). Moreover, we recently found that Pex5p, using PEX5-defective ZP105 and ZP139. Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD. Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126.     

Quantitative analysis of peroxisomal targeting signal type-1 binding to wild-type and pathogenic mutants of Pex5p supports an affinity threshold for peroxisomal protein targeting

Peroxisomal biogenesis disorders (PBDs) are caused by mutations in 12 distinct genes that encode the components of the peroxisome assembly machinery. Three mutations in the gene encoding Pex5p, the peroxisomal targeting signal type-1 (PTS1) receptor, have been reported, each associated with a disorder of the Zellweger spectrum of different severity. Here, we report studies of the affinities of mutated forms of Pex5p for a series of PTS1 peptides and conclude that PTS1-affinity reductions are correlated with disease severity and cell biological phenotype. A quantitative model has been developed that allows estimation of the dissociation constants for complexes with a wide range of PTS1 sequences bound to wild-type and mutant Pex5p. In the context of this model, the binding measurements suggest that no PTS1-containing proteins are targeted by Pex5p(N489K) and only a relatively small subset of PTS1-containing proteins with the highest affinity for Pex5p are targeted to peroxisomes by Pex5p(S563W). Furthermore, the results of the analysis are consistent with an approximate dissociation constant threshold near 500 nM required for efficient protein targeting to peroxisomes.     

Synthesis of a heterotrifunctional linker for the site-specific preparation of antibody-drug conjugates with two distinct warheads

Codelivery of multiple therapeutic agents with different anticancer mechanisms can overcome drug resistance as well as generate additive or synergistic anticancer effects that may enhance the antitumor efficacy. Antibody-drug conjugates (ADCs) can be used for highly specific delivery of multiple therapeutic agents with different anticancer mechanisms, though more research is required towards designing flexible platforms on which dual drug ADCs could be prepared. Herein, we describe the synthesis of a heterotrifunctional linker that could be used to construct flexible platforms for preparing dual-cytotoxic drug conjugates in a site-specific manner. As a proof of concept, we synthesized dual drug ADCs carrying monomethyl auristain E (MMAE, tubulin polymerization inhibitor) and pyrrolobenzodiazepine dimer (PBD, DNA minor groove alkylator). We then evaluated the dual drug ADCs for in vitro efficacy and confirmed the dual mechanism of action.     

Synthesis of a novel C2/C2′-aryl-substituted pyrrolo[2,1-c][1,4]benzodiazepine dimer prodrug with improved water solubility and reduced DNA reaction rate

A prodrug form (17) of a novel C2/C2′-aryl-substituted pyrrolobenzodiazepine (PBD) dimer (16) has been synthesized by introducing sodium bisulfite groups to the C11/C11′-positions of the parent bis-imine. The prodrug form is highly water soluble, stable in aqueous conditions, and the rate of DNA cross-link formation is much slower compared to the parent bis-imine.     

Temperature sensitivity in peroxisome assembly processes characterizes milder forms of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) contain various clinical phenotypes; Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), decreasing in the clinical severity in this order. We found that all IRD cell lines and some NALD lines belonging to several different complementation groups are temperature-sensitive in peroxisome assembly; that is, they lacked catalase-positive peroxisomes at 37°C, but do gain the peroxisomes at 30°C. We identified heterozygous mutations E55K/R119Stop in the PEX2 gene of an IRD patient of complementation group F. The E55K mutation was the direct cause of the temperature-sensitivity because similar phenotypes could be transferred to PEX2-defective CHO cells by transfecting the mutant gene. Thus, temperature-sensitive peroxisome assembly is representative of milder forms of PBDs. The main part of this study was published by Imamura et al. (1).