PABPN1 gene therapy for oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles.

All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation.

Metallothionein (MT) is a small cysteine-rich protein thought to play a critical role in cellular detoxification of inorganic species by sequestering metal ions that are present in elevated concentrations. We demonstrate here that metallothionein can play an important role at the other end of the homeostatic spectrum by scavenging an essential metal in a mouse fibroblast cell line that has been cultured under conditions of extreme zinc deprivation (LZA-LTK-). These cells unexpectedly produce constitutively high levels of metallothionein mRNA; however, the MT protein accumulates only when high concentrations of zinc are provided in the media. Until this MT pool is saturated, no measurable zinc remains in the external media. In this case, zinc deprivation leads to amplification of the MT gene locus in the LZA-LTK- cell line. Furthermore, the intracellular zinc levels in the fully adapted cells remain at the normal level of 0.4 fmol zinc/cell, even when extracellular zinc concentration is decreased by 2 orders of magnitude relative to normal media.

The chemical cell biology of zinc: structure and intracellular fluorescence of a zinc-quinolinesulfonamide complex.

Fluorescent cell-permeant compounds based on 6-methoxy-8-p-toluenesulfonamido-quinoline, TSQ, are potentially powerful probes of intracellular zinc chemistry; however, the structure, thermodynamics, and stoichiometry of the metal complexes, and the molecular basis of Zn(II) recognition, remain open issues. To address these, we report the first structural characterization of a Zn(II) complex of a TSQ derivative, namely 2-methyl-6-methoxy-8-p-toluenesulfonamido-quinoline (3) and describe its unusual coordination chemistry. The crystal structure of the fluorescent complex of 3 with zinc reveals a 2:1 stoichiometry wherein bidentate coordination of two nitrogens from each ligand gives rise to a highly distorted tetrahedral Zn(II) center. Both sulfonamido groups in the zinc complex are tilted away from zinc to make room for coordination of the amide nitrogens. Zn-O(2) and Zn-O(4) distances are essentially nonbonding (3.06 and 3.10 A, respectively). The bond angles [N(1)-Zn-N(2) 83.5 degrees and N(3)-Zn-N(4) 83.0 degrees] are quite small relative to the 109 degrees angle of an ideal tetrahedral center. This result provides an insight into the zinc-binding mode of the TSQ derivative zinquin, in which a methyl group replaces the hydrogen in the 2-position of the quinoline ring. The methyl group and sulfonamide oxygen atoms clearly hinder formation of both square planar and octahedral complexes. We also show here that the Zn(II) complex of 3 in DMSO-water (80/20 w/w) exhibits an overall binding stability (log beta 2 = 18.24 +/- 0.02) similar to zinquin. Fluorescence microscopy suggests that each of these members of this family demarks a similar set of Zn(II)-enriched compartments that are common to all eukaryotic cells examined to date, and further shows that the ester function is not required for observation of these ubiquitous Zn-loaded compartments. The combined structural, thermodynamic, and physiological results provide a basis for design of other Zn(II)-specific membrane permeant probes with a range of Zn(II) affinities and photophysical properties.