There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immunecompromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and in the face of osmotic stress. These studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The principal mechanism by which yeast cells detect and respond to cell wall stress is through the Cell Wall Integrity (CWI) signaling pathway, which transmits stress signals generated at the cell surface to a GTPase switch that activates a MAP kinase cascade. However, there is an additional pathway that contributes to the structural integrity of the cell wall in response to hypo-osmotic shock. This pathway culminates in the reduction of turgor pressure by release of intracellular glycerol through the Fps1 glycerol channel.
The specific aims of this project are 1) To determine the function of the Mpk1 cell wall stress MAP kinase when bound to the Paf1-RNA polymerase transcription elongation complex (PafC). Recent discoveries have revealed that Mpk1 activates a subset of its transcriptional program through a non-catalytic mechanism that involves its association with the promoters and coding regions of its transcriptional targets. New data suggests that Mpk1 moves from the promoter to the PafC. Experiments are described to dissect the function of Mpk1 within the context of this complex. 2) To establish the nature of the relationship between CWI signaling and DNA damage checkpoint signaling. Mpk1 is activated in reponse to DNA damaging agents, but its cell wall transcriptional program is not activated under these conditions. It is hypothesized that novel serine phosphorylations on Mpk1 provoked by DNA damage checkpoint kinases redirect it from cell wall targets to other functions relevant to the DNA damage response. Experiments are proposed to test this hypothesis and to identify the role of Mpk1 in this response. 3) To establish the mechanism by which a pair of novel regulators of the Fps1 glycerol channel, Rgc1 and Rgc2, activate Fps1 and to identify regulatory phosphorylation sites on these proteins in response to various stress signals. Preliminary data suggest that Rgc1/2 serve as regulatory nodes for multiple protein kinases. Experiments are proposed to identify pathway components both upstream and downstream of Rgc1/2. 4) To determine if the Pkc1 protein kinase of the CWI pathway contributes to the G2/M transition through the RSC chromatin remodeling complex. A recently-discovered physical interaction between Pkc1 and the Rsc1 subunit of the RSC complex suggests a mechanism by which Pkc1 contributes to the G2/M transition. Experiments are proposed to test this possibility.

Public Health Relevance

There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immune-compromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. Proposed studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM048533-21S1
Application #
8729150
Study Section
Program Officer
Reddy, Michael K
Project Start
1992-09-30
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
21
Fiscal Year
2014
Total Cost
$37,106
Indirect Cost
$14,439
Name
Boston University
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Beese-Sims, Sara E; Lee, Jongmin; Levin, David E (2011) Yeast Fps1 glycerol facilitator functions as a homotetramer. Yeast 28:815-9
Levin, David E (2011) Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics 189:1145-75
Kim, Ki-Young; Levin, David E (2011) Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination. Cell 144:745-56
Kim, Ki-Young; Levin, David E (2010) Transcriptional reporters for genes activated by cell wall stress through a non-catalytic mechanism involving Mpk1 and SBF. Yeast 27:541-8
Kim, Ki-Young; Truman, Andrew W; Caesar, Stefanie et al. (2010) Yeast Mpk1 cell wall integrity mitogen-activated protein kinase regulates nucleocytoplasmic shuttling of the Swi6 transcriptional regulator. Mol Biol Cell 21:1609-19
Koutmos, Markos; Kabil, Omer; Smith, Janet L et al. (2010) Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine {beta}-synthase. Proc Natl Acad Sci U S A 107:20958-63
Beese, Sara E; Negishi, Takahiro; Levin, David E (2009) Identification of positive regulators of the yeast fps1 glycerol channel. PLoS Genet 5:e1000738
Truman, Andrew W; Kim, Ki-Young; Levin, David E (2009) Mechanism of Mpk1 mitogen-activated protein kinase binding to the Swi4 transcription factor and its regulation by a novel caffeine-induced phosphorylation. Mol Cell Biol 29:6449-61
Matthews, Rowena G; Koutmos, Markos; Datta, Supratim (2008) Cobalamin-dependent and cobamide-dependent methyltransferases. Curr Opin Struct Biol 18:658-66
Kim, Ki-Young; Truman, Andrew W; Levin, David E (2008) Yeast Mpk1 mitogen-activated protein kinase activates transcription through Swi4/Swi6 by a noncatalytic mechanism that requires upstream signal. Mol Cell Biol 28:2579-89

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