Molecular and cellular effects of Tamm-Horsfall protein mutations and their rescue by chemical chaperones

J Biol Chem. 2012 Jan 6;287(2):1290-305. doi: 10.1074/jbc.M111.283036. Epub 2011 Nov 22.

Abstract

Correct folding of a nascent polypeptide in the lumen of the endoplasmic reticulum (ER) into a three-dimensional conformation is a crucial step in the stability, intracellular trafficking, and targeting to the final destination of a protein. By transiently and stably expressing human-relevant mutants of Tamm-Horsfall protein in polarized Madin-Darby canine kidney cells, we show here that a cysteine-altering mutation in the evolutionally conserved cysteine-rich domain had more severe defects in ER exit and surface translocation and triggered more apoptosis than a cysteine-altering mutation outside the domain. Both mutants were able to specifically bind and trap the wild-type Tamm-Horsfall protein (THP) and prevent it from exiting the ER and translocating to the cell surface. This explains at least partly why in patients with THP-associated diseases there is a marked urinary reduction of both the mutant and the wild-type THP. Exposure of mutant-expressing cells to low temperature (30 °C), osmolytes (glycerol, trimethylamine N-oxide, and dimethyl sulfoxide), and the Ca(2+)-ATP inhibitor thapsigargin only slightly relieved ER retention and increased surface targeting of the mutants. In contrast, sodium 4-phenylbutyrate and probenecid, the latter a uricosuric drug used clinically to treat gout, markedly reduced ER retention of the mutants and increased their surface translocation and secretion into the culture media. The rescue of the THP mutants was associated with the restoration of the level and subcellular localization of cytosolic chaperone HSP70. Our results reveal intricate mechanistic details that may underlie THP-associated diseases and suggest that novel therapeutics enhancing the refolding of THP mutants may be of important value in therapy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Apoptosis / genetics
  • Cell Line
  • Dogs
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum / pathology
  • Genetic Diseases, Inborn / genetics
  • Genetic Diseases, Inborn / metabolism
  • Genetic Diseases, Inborn / pathology
  • Genetic Diseases, Inborn / therapy
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism*
  • Humans
  • Kidney Diseases, Cystic / genetics
  • Kidney Diseases, Cystic / metabolism
  • Kidney Diseases, Cystic / pathology
  • Kidney Diseases, Cystic / therapy
  • Mice
  • Mutation*
  • Polycystic Kidney, Autosomal Dominant
  • Protein Folding*
  • Protein Structure, Tertiary
  • Protein Transport / genetics
  • Uromodulin / genetics
  • Uromodulin / metabolism*

Substances

  • HSP70 Heat-Shock Proteins
  • UMOD protein, human
  • Umod protein, mouse
  • Uromodulin

Supplementary concepts

  • Medullary Cystic Kidney Disease 2