Rapid purification of active gamma-secretase, an intramembrane protease implicated in Alzheimer's disease

γ-Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-β precursor protein generates the amyloid-β protein (Aβ) of Alzheimer's disease, partially inhibiting γ-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ-secretase, but at low resolution (15 Å). The limited amount of purified γ-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four γ-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active γ-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.     

Environment of the active site region of RseP, an Escherichia coli regulated intramembrane proteolysis protease, assessed by site-directed cysteine alkylation

Regulated intramembrane proteolysis (RIP) plays crucial roles in both prokaryotic and eukaryotic organisms. Proteases for RIP cleave transmembrane regions of substrate membrane proteins. However, the molecular mechanisms for the proteolysis of membrane-embedded transmembrane sequences are largely unknown. Here we studied the environment surrounding the active site region of RseP, an Escherichia coli S2P ortholog involved in the sigma(E) pathway of extracytoplasmic stress responses. RseP has two presumed active site motifs, HEXXH and LDG, located in membrane-cytoplasm boundary regions. We examined the reactivity of cysteine residues introduced within or in the vicinity of these two active site motifs with membrane-impermeable thiol-alkylating reagents under various conditions. The active site positions were inaccessible to the reagents in the native state, but many of them became partially modifiable in the presence of a chaotrope, while requiring simultaneous addition of a chaotrope and a detergent for full modification. These results suggest that the active site of RseP is not totally embedded in the lipid phase but located within a proteinaceous structure that is partially exposed to the aqueous milieu.     

Determination of the cleavage sites in SulA, a cell division inhibitor, by the ATP-dependent HslVU protease from Escherichia coli

HslVU is an ATP-dependent protease from Escherichia coli and known to degrade SulA, a cell division inhibitor, both in vivo and in vitro, like the ATP-dependent protease Lon. In this study, the cleavage specificity of HslVU toward SulA was investigated. The enzyme was shown to produce 58 peptides with various sizes (3-31 residues), not following the 'molecular ruler' model. Cleavage occurred at 39 peptide bonds preferentially after Leu in an ATP-dependent manner and in a processive fashion. Interestingly, the central and C-terminal regions of SulA, which are known to be important for the function of SulA, such as inhibition of cell division and molecular interaction with certain other proteins, were shown to be preferentially cleaved by HslVU, as well as by Lon, despite the fact that the peptide bond specificities of the two enzymes were distinct from each other.     

Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp. SP1(1)

Aims:  Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach.
Methods and Results:  The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl₃. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l⁻¹): NaCl, 250; KCl, 2; MgSO₄, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl₃, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml⁻¹ of protease.
Conclusions:  Soybean flour and FeCl₃ were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production.
Significance and Impact of the Study:  The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.     

Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.     

Decrease in cell viability in an RMF,sigma(38), and OmpC triple mutant of Escherichia coli

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.     

Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA.

We have isolated three multicopy suppressors of the conditional lethal phenotype of a prc (tsp) null strain of Escherichia coli. One of these suppressors included two novel putative protease genes in tandem that map to 3400 kb or 72.5 centisomes on the chromosome. We propose the names hhoA and hhoB, for htrA homolog, to denote that these genes encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease and 36% identical to each other. The HhoA and HhoB proteins are predicted to be 455 and 355 amino acids, respectively, in length. The mature HhoA protein is periplasmic in location, and amino-terminal sequencing shows that it arises following cleavage of a 27-amino-acid signal peptide. Searches of the protein and DNA databases reveal a rapidly growing family of homologous genes in a variety of other bacteria, including several which are required for virulence in their host. Deletion of the hhoAB genes shows that they are not required for viability at high temperatures like the homologous htrA but grow more slowly than wild-type strains. A second multicopy prc suppressor is the dksA (dnaK suppressor) gene, which is also a multicopy suppressor of defects in the heat shock genes dnaK, dnaJ, and grpE. The dksA gene was independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. A third dosage-dependent prc suppressor includes a truncated rare lipoprotein A (rlpA) gene.     

Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response

CYP19 (P450arom) catalyzes the aromatization reaction of C19 steroids leading to estrogens. While readily expressed in insect cells, the human P450arom has been a difficult P450 to express in Escherichia coli at useful levels. In the present study, we replaced the N-terminal sequence in human CYP19 with the corresponding sequences of other microsomal P450s (CYP2C11 and CYP17) that are efficiently expressed in E. coli. Although the N-terminal replacement alone was not sufficient for the expression, human P450arom was successfully expressed up to the level of 240nmol/l culture by the combination of the N-terminal replacement and the induction of cold stress response by 1 microg/ml chloramphenicol. Membrane fractions containing the expressed P450arom catalyzed aromatization of androstenedione with a specific activity of 4.9 nmol/min/nmol P450. Our results are important to provide large quantities of human P450arom as an active form for structure-function studies.     

ZitB (YbgR), a Member of the Cation Diffusion Facilitator Family, Is an Additional Zinc Transporter in Escherichia coli

The Escherichia coli zitB gene encodes a Zn(II) transporter belonging to the cation diffusion facilitator family. ZitB is specifically induced by zinc. ZitB expression on a plasmid rendered zntA-disrupted E. coli cells more resistant to zinc, and the cells exhibited reduced accumulation of (65)Zn, suggesting ZitB-mediated efflux of zinc.     

Overexpression of fetA (ybbL) and fetB (ybbM), Encoding an Iron Exporter,Enhances Resistance to Oxidative Stress in Escherichia coli

Reactive oxygen species are generated by redox reactions and the Fenton reaction of H₂O₂ and iron that generates the hydroxyl radical that causes severe DNA, protein, and lipid damage. We screened Escherichia coli genomic libraries to identify a fragment, containing cueR, ybbJ, qmcA, ybbL, and ybbM, which enhanced resistance to H₂O₂ stress. We report that the ΔybbL and ΔybbM strains are more susceptible to H₂O₂ stress than the parent strain and that ybbL and ybbM overexpression overcomes H₂O₂ sensitivity. The ybbL and ybbM genes are predicted to code for an ATP-binding cassette metal transporter, and we demonstrate that YbbM is a membrane protein. We investigated various metals to identify iron as the likely substrate of this transporter. We propose the gene names fetA and fetB (for Fe transport) and the gene product names FetA and FetB. FetAB allows for increased resistance to oxidative stress in the presence of iron, revealing a role in iron homeostasis. We show that iron overload coupled with H₂O₂ stress is abrogated by fetA and fetB overexpression in the parent strain and in the Δfur strain, where iron uptake is deregulated. Furthermore, we utilized whole-cell electron paramagnetic resonance to show that intracellular iron levels in the Δfur strain are decreased by 37% by fetA and fetB overexpression. Combined, these findings show that fetA and fetB encode an iron exporter that has a role in enhancing resistance to H₂O₂-mediated oxidative stress and can minimize oxidative stress under conditions of iron overload and suggest that FetAB facilitates iron homeostasis to decrease oxidative stress.     

Osmoregulation of the HtrA (DegP) protease of Escherichia coli: an Hha-H-NS complex represses HtrA expression at low osmolarity

The HtrA protein of Escherichia coli is a heat-shock inducible periplasmic protease, essential for bacterial survival at high temperatures. Expression of htrA gene depends on the alternative factor sigmaE and on the two-component regulatory system Cpx. These regulators systems respond, among others factors, to overproduction of misfolded proteins in the periplasm or to high level synthesis of various extracytoplasmic proteins. We describe in this report the osmoregulation of the expression of htrA gene. Low osmolarity conditions result in htrA repression. We report, as well, the role of the nucleoid associated proteins H-NS and Hha in the repression of htrA expression at low osmolarity.     

Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review

Dps, the DNA‐binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The structure and function of Dps have been the subject of numerous studies and have been examined in several bacteria that possess Dps or a structural/functional homologue of the protein. Additionally, the involvement of Dps in stress resistance has been researched extensively as well. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. Regulation of Dps expression in E. coli is complex and partially dependent on the physiological state of the cell. Furthermore, it is proposed that Dps itself plays a role in gene regulation during starvation, ultimately making the cell more resistant to cytotoxic assaults by controlling the expression of genes necessary for (or deleterious to) stress resistance. The current review focuses on the aforementioned properties of Dps in E. coli, its prototypic organism. The consequences of elucidating the protective mechanisms of this protein are far‐reaching, as Dps homologues have been identified in over 1000 distantly related bacteria and Archaea. Moreover, the prevalence of Dps and Dps‐like proteins in bacteria suggests that protection involving DNA and iron sequestration is crucial and widespread in prokaryotes.