Final Activity Report Summary - HOTPROT (Heat shock protein 70 and the cytosolic sensing of protein accumulation)
Plants have to cope with a range of abiotic and biotic stresses such as high temperature and invasive diseases. Both of these examples are associated with the accumulation of misfolded proteins, which is managed by the plant through the induction of protein chaperones to assist with protein refolding.
We firstly observed this in plant cells infected with viruses which accumulated large amounts of viral proteins in the cell cytoplasms and induced a specific range of chaperones called cytosolic HSP70s1. Plant and animal responses to misfolded protein in a specific subcompartment of cells, the endoplasmic reticulum (ER), were studied extensively before, but responses to the accumulation of misfolded protein in the soluble cytosol, i.e. outside of the ER, was a novel area of research. The former process was called the unfolded protein response (UPR). We named this new phenomenon the cytosolic protein response (CPR) and compared the underlying mechanisms with those for the UPR.
To consistently induce the CPR, the accumulation of misfolded proteins was induced by treatment with a chemical azetidine-2-carboxylic acid (AZC). This was very similar to the amino acid proline and was used instead of it to make proteins. However, due to a chemical structural difference, AZC created misfolded proteins. To understand some of the underlying mechanisms, AZC treated leaves were examined for changes in the expression of all genes using a technology called microarray analysis. Since AZC induced misfolded protein accumulation in both ER and cytosol, effects were compared with leaves treated with another chemical (tunicamycin) that specifically induced the UPR.
A range of proteins called heat shock transcription factors regulated the expression of HSP70 genes and were therefore potential regulators of the CPR. One particular factor, HSFA2 and a novel, alternatively processed or spliced' form, HSFA2-AZC, were strongly induced during the CPR after treatment with AZC. The same effect was seen after virus infection, for the treatment of which the original observations were made. Hence, HSFA2-AZC appeared to be a signature for the CPR. HSFA2 activity induced the expression of the HSP70 chaperones, probably through an interaction with a specific piece of deoxyribonucleic acid (DNA) known as the heat shock element, HSE. Interestingly, over-expression of HSFA2 increased the tolerance of plant to AZC. From sequence analysis, the HSFA2-AZC form was likely not to have an active function. It was proposed that alternate splicing of HSFA2 might be one way of controlling the amount of active HSFA2 during the response.
If the induction of HSFA2-AZC were a signature of the CPR and one consequence of heat stress was protein misfolding, then heat stress should also induce HSFA2-AZC. The total gene expression profile of heat-shocked plants showed the induction of HSFA2 and HSFA2-AZC, confirming that the heat shock response involved the CPR. By comparing microarray analyses for arabidopsis plants treated with heat, AZC and tunicamycin we hoped to identify the molecular pathways involved in the CPR. We expected that responses to heat-treatment and AZC treatment would both include the CPR and UPR and that response to tunicamycin would be restricted to the UPR. We identified 904 genes that were affected by AZC but not by tunicamycin, i.e. were CPR specific. From these, 425 genes were also differentially expressed after heat shock. These genes, which potentially identified the CPR subcomponent of the heat shock response included transcription factors, various chaperones, splicing factors and components of the protein degradation machinery.
Therefore, we identified one of the controllers of the CPR, HSFA2, the DNA elements required for the CPR and genes differentially expressed during the CPR. Our study revealed the previously uncharacterised process and the mechanisms of CPR.
We firstly observed this in plant cells infected with viruses which accumulated large amounts of viral proteins in the cell cytoplasms and induced a specific range of chaperones called cytosolic HSP70s1. Plant and animal responses to misfolded protein in a specific subcompartment of cells, the endoplasmic reticulum (ER), were studied extensively before, but responses to the accumulation of misfolded protein in the soluble cytosol, i.e. outside of the ER, was a novel area of research. The former process was called the unfolded protein response (UPR). We named this new phenomenon the cytosolic protein response (CPR) and compared the underlying mechanisms with those for the UPR.
To consistently induce the CPR, the accumulation of misfolded proteins was induced by treatment with a chemical azetidine-2-carboxylic acid (AZC). This was very similar to the amino acid proline and was used instead of it to make proteins. However, due to a chemical structural difference, AZC created misfolded proteins. To understand some of the underlying mechanisms, AZC treated leaves were examined for changes in the expression of all genes using a technology called microarray analysis. Since AZC induced misfolded protein accumulation in both ER and cytosol, effects were compared with leaves treated with another chemical (tunicamycin) that specifically induced the UPR.
A range of proteins called heat shock transcription factors regulated the expression of HSP70 genes and were therefore potential regulators of the CPR. One particular factor, HSFA2 and a novel, alternatively processed or spliced' form, HSFA2-AZC, were strongly induced during the CPR after treatment with AZC. The same effect was seen after virus infection, for the treatment of which the original observations were made. Hence, HSFA2-AZC appeared to be a signature for the CPR. HSFA2 activity induced the expression of the HSP70 chaperones, probably through an interaction with a specific piece of deoxyribonucleic acid (DNA) known as the heat shock element, HSE. Interestingly, over-expression of HSFA2 increased the tolerance of plant to AZC. From sequence analysis, the HSFA2-AZC form was likely not to have an active function. It was proposed that alternate splicing of HSFA2 might be one way of controlling the amount of active HSFA2 during the response.
If the induction of HSFA2-AZC were a signature of the CPR and one consequence of heat stress was protein misfolding, then heat stress should also induce HSFA2-AZC. The total gene expression profile of heat-shocked plants showed the induction of HSFA2 and HSFA2-AZC, confirming that the heat shock response involved the CPR. By comparing microarray analyses for arabidopsis plants treated with heat, AZC and tunicamycin we hoped to identify the molecular pathways involved in the CPR. We expected that responses to heat-treatment and AZC treatment would both include the CPR and UPR and that response to tunicamycin would be restricted to the UPR. We identified 904 genes that were affected by AZC but not by tunicamycin, i.e. were CPR specific. From these, 425 genes were also differentially expressed after heat shock. These genes, which potentially identified the CPR subcomponent of the heat shock response included transcription factors, various chaperones, splicing factors and components of the protein degradation machinery.
Therefore, we identified one of the controllers of the CPR, HSFA2, the DNA elements required for the CPR and genes differentially expressed during the CPR. Our study revealed the previously uncharacterised process and the mechanisms of CPR.