Stanley M. Stevens

Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1

Heat shock factor 1 (HSF1) is essential for protecting cells from protein-damaging stress associated with misfolded proteins and regulates the insulin-signaling pathway and aging. Here, we show that human HSF1 is inducibly acetylated at a critical residue that negatively regulates DNA binding activity. Activation of the deacetylase and longevity factor SIRT1 prolonged HSF1 binding to the heat shock promoter Hsp70 by maintaining HSF1 in a deacetylated, DNA–binding competent state. Conversely, down-regulation of SIRT1 accelerated the attenuation of the heat shock response (HSR) and release of HSF1 from its cognate promoter elements. These results provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and the HSR.

Proteomic analysis of the synaptic plasma membrane fraction isolated from rat forebrain.

Mass spectrometry (MS) in conjunction with liquid chromatography and gel separation techniques has been utilized to identify synaptic plasma membrane (SPM) proteins isolated from rat forebrain and digested with the protease trypsin. Initial results employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation of the SPM protein mixture have shown that several membrane proteins were under-represented due to solubilization problems in the dimension of isoelectric-point focusing. Given the complexity of the SPM, multiple stages of separation were necessary prior to mass spectrometric detection in order to facilitate protein identification. This particular study involved several approaches using one-dimensional (1D) sodium dodecyl sulfate (SDS)-PAGE, strong cation-exchange (SCX) chromatography and capillary reversed-phase high performance liquid chromatography (HPLC) techniques. In addition to these gel and HPLC separation stages, complementary information was obtained by using both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Data-dependent acquisition employing capillary HPLC-nanoESI/MS allowed for the detection of low-abundance tryptic peptides in the digested SPM fraction and identification of the corresponding proteins when product-ion information of a single or multiple peptides was used in protein database searching. The potential value of this subproteome methodology was exemplified by the identification of several proteins relevant to synaptic physiology which included various transporters, receptors, ion channels, and enzymes.

Cytosolic proteins lose solubility as amyloid deposits in a transgenic mouse model of Alzheimer-type amyloidosis

The extracellular accumulation of β-amyloid peptide is a key trigger in the pathogenesis of Alzheimers disease (AD). In humans, amyloid deposition precedes the appearance of intracellular inclusion pathology formed by cytosolic proteins such as Tau, α-synuclein and TDP-43. These secondary pathologies have not been observed in mice that model Alzheimer-type amyloidosis by expressing mutant amyloid precursor protein, with or without mutant presenilin 1. The lack of secondary pathology in these models has made it difficult to establish how amyloid deposition initiates the cascade of events that leads to secondary intracellular pathology that characterizes human AD. In transgenic mice that model Alzheimer-type amyloidosis, we sought to determine whether there is evidence of altered cytosolic protein folding by assessing whether amyloid deposition causes normally soluble proteins to misfold. Using a method that involved detergent extraction and sedimentation coupled with proteomic approaches, we identified numerous cytosolic proteins that show specific losses in solubility as amyloid accumulates. The proteins identified included glycolytic enzymes and members of the 14-3-3 chaperone family. A substantial accumulation of lysine 48-linked polyubiquitin was also detected. Overall, the data demonstrate that the accumulation of amyloid by some manner causes the loss of solubility intracellular cytosolic proteins.

A phosphorylation map of the bovine papillomavirus E1 helicase

BackgroundPapillomaviruses undergo a complex life cycle requiring regulated DNA replication. The papillomavirus E1 helicase is essential for viral DNA replication and plays a key role in controlling viral genome copy number. The E1 helicase is regulated at least in part by protein phosphorylation, however no systematic approach to phosphate site mapping has been attempted. We have utilized mass spectrometry of purified bovine papillomavirus E1 protein to identify and characterize new sites of phosphorylation.ResultsMass spectrometry and in silico sequence analysis were used to identify phosphate sites on the BPV E1 protein and kinases that may recognize these sites. Five new and two previously known phosphorylation sites were identified. A phosphate site map was created and used to develop a general model for the role of phosphorylation in E1 function.ConclusionMass spectrometric analysis identified seven phosphorylated amino acids on the BPV E1 protein. Taken with three previously identified sites, there are at least ten phosphoamino acids on BPV E1. A number of kinases were identified by sequence analysis that could potentially phosphorylate E1 at the identified positions. Several of these kinases have known roles in regulating cell cycle progression. A BPV E1 phosphate map and a discussion of the possible role of phosphorylation in E1 function are presented.

Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.

The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.