The Effects of Molecular Chaperone Modulation on Protein Folding, Prion Formation, and Prion Propagation in Saccharomyces cerevisiae

Proper and efficient protein folding is vital for cell survival. Many factors affect protein folding fidelity and prion formation, including molecular chaperone availability and activity. Research has shown that modulating chaperone availability and function can affect protein misfolding and aggregation, as well as de novo prion formation and propagation. However, the factors involved and underlying mechanisms influencing prion formation and protein folding are largely unknown. The following work aims to elucidate these areas. The Nascent Polypeptide-Associated Complex (NAC) is the first point of chaperone contact for nascent polypeptides. Previous work has shown that disruption of the NAC leads to improved viability in cells experiencing protein misfolding stress. This counterintuitive result led us to investigate the ability of NAC deletion to improve survivability of cells expressing misfolding human proteins. This work resulted in the identification of multiple NAC deletion strains that improve viability in cells expressing disease-causing alpha-synuclein and expanded polyglutamine proteins. Also, this work identified changes in de novo induction of a yeast prion and morphological changes in expanded polyglutamine aggregates as a result of NAC disruption. Overall, this work reveals the potential of NAC disruption as a therapeutic target for neurodegenerative diseases and sets the stage for investigating the mechanism by which NAC disruption improves viability in cells expressing disease-causing, aggregating proteins. Mutations in another chaperone, DNAJB6, have been shown to cause Limb-Girdle Muscular Dystrophy Type 1D (LGMDD1). While we know that these mutations are associated with LGMDD1, the mechanism by which they induce disease remains unknown. Because substrates of DNAJB6 have not been identified, we have turned to a homologous protein in yeast, Sis1, with known client proteins to better understand the effect of these mutations. We have also developed a Sis1-DNAJB6 chimeric protein (SDSS) to evaluate these mutations. This chimeric protein includes the J, G/M, and C-terminal domains of Sis1, and the G/F domain, in which many LGMDD1-associated mutations are found, of DNAJB6. Previous work has shown that when LGMDD1-associated mutations are introduced in Sis1 or SDSS there is disruption of client processing by Sis1. This body of work identifies multiple second-site suppressors that, when introduced in combination with LGMDD1-associated mutations, are capable of recovering client processing by Sis1 and SDSS. Overall, this work shows that second-site suppressors may be capable of recovering DNAJB6 activity when introduced in combination with LGMDD1-associated mutations. Moreover, it provides an experimental model for the continued investigation of these second-site suppressors and identification of similar therapeutic avenues for potentially treating patients with other LGMDD1-associated mutations in the future

The zinc finger transcription factor PLAGL2 enhances stem cell fate and activates expression of ASCL2 in intestinal epithelial cells

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The Zinc Finger Transcription Factor PLAGL2 Enhances Stem Cell Fate and Activates Expression of ASCL2 in Intestinal Epithelial Cells

Summary: Intestinal epithelial stem cell (IESC) fate is promoted by two major transcriptional regulators, the TCF4/β-catenin complex and ASCL2, which drive expression of IESC-specific factors, including Lgr5, Ephb2, and Rnf43. Canonical Wnt signaling via TCF4/β-catenin directly transactivates Ascl2, which in turn auto-regulates its own expression. Conversely, Let-7 microRNAs antagonize the IESC lineage by repressing specific mRNA targets. Here, we identify the zinc finger transcription factor PLAGL2 as a Let-7 target that regulates IESC fate. PLAGL2 drives an IESC expression signature, activates Wnt gene expression, and enhances a TCF/LEF reporter in intestinal organoids. In parallel, via cell-autonomous mechanisms, PLAGL2 is required for lineage clonal expansion and directly enhances expression of ASCL2. PLAGL2 also supports enteroid growth and survival in the context of Wnt ligand depletion. PLAGL2 expression is strongly associated with an IESC signature in colorectal cancer and may be responsible for contributing to the aberrant activation of an immature phenotype. : In this article, Madison and colleagues show that the zinc finger transcription factor PLAGL2 is a potent driver of intestinal stem cell lineage specification in organoids. This is mediated both via secreted signals, likely Wnts, activated by PLAGL2, and also by cell-autonomous and direct PLAGL2 activation of Ascl2, a transcription factor that drives stem cell fate. Keywords: Let-7, PLAGL2, ASCL2, intestinal epithelium, stem cel