Correcting the F508del-CFTR variant by modulating eukaryotic translation initiation factor 3-mediated translation initiation.

Inherited and somatic rare diseases result from >200,000 genetic variants leading to loss- or gain-of-toxic function, often caused by protein misfolding. Many of these misfolded variants fail to properly interact with other proteins. Understanding the link between factors mediating the transcription, translation, and protein folding of these disease-associated variants remains a major challenge in cell biology.

Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy.

The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants.

Using Histone Deacetylase Inhibitors to Analyze the Relevance of HDACs for Translation.

Gene expression is regulated in part through the reversible acetylation of histones, by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). HAT activity results in the addition of acetyl groups on the lysine residues of histone tails leading to decondensation of the chromatin, and increased gene transcription in general, whereas HDACs remove these acetyl groups, thus leading to an overall suppression of gene transcription. Recent evidence has elucidated that histones are not the only components of the proteome that are targeted by HATs and HDACs.

Modulation of the maladaptive stress response to manage diseases of protein folding.

Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer's disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive.

The histone deacetylase inhibitor, Vorinostat, represses hypoxia inducible factor 1 alpha expression through translational inhibition.

Hypoxia inducible factor 1α (HIF-1α) is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi) block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA) and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma.

Q-bodies monitor the quinary state of the protein fold.

Cytoplasmic compartments containing misfolded proteins targeted for degradation, named Q-bodies, have been identified. Q-body formation is a dynamic process that actively manages the metastable state of the protein fold through small heat shock proteins and the Hsp70-Hsp90-Hsp110 proteostasis system to promote cellular fitness under both physiological and stress conditions.

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability.

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell.

Mechanism of microtubule-facilitated "fast track" nuclear import.

Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold.

The flexible loop of the human cytomegalovirus DNA polymerase processivity factor ppUL44 is required for efficient DNA binding and replication in cells.

Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP(174)]), which has been proposed to be directly involved in ppUL44's interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Deltaloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids.

Dynein light chain association sequences can facilitate nuclear protein import.

Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures.

A microtubule-facilitated nuclear import pathway for cancer regulatory proteins.

Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time.