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1.  Endoplasmic Reticulum-Dependent Redox Reactions Control Endoplasmic Reticulum-Associated Degradation and Pathogen Entry 
Antioxidants & Redox Signaling  2012;16(8):809-818.
Abstract
Significance Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host–pathogen interactions. Recent Advances: Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. Critical Issues: The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. Future Directions: How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions. Antioxid. Redox Signal. 16, 809–818.
doi:10.1089/ars.2011.4425
PMCID: PMC3283440  PMID: 22142231
2.  High Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume By Exploiting Energy Transfer 
Journal of biomolecular screening  2010;15(10):1211-1219.
Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate-detection reagents, such as quinaldine red (QR). While such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high throughput screening. To overcome these difficulties, we have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal. Biochem. 2005, 342:254–259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~8%) and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70-family members.
doi:10.1177/1087057110380571
PMCID: PMC3052282  PMID: 20926844
phosphate; malachite green; ATPase; molecular chaperone; fluorescence assay
3.  A PDI Family Network Acts Distinctly and Coordinately with ERp29 To Facilitate Polyomavirus Infection▿  
Journal of Virology  2010;85(5):2386-2396.
Endoplasmic reticulum (ER)-to-cytosol membrane transport is a decisive infection step for the murine polyomavirus (Py). We previously determined that ERp29, a protein disulfide isomerase (PDI) member, extrudes the Py VP1 C-terminal arm to initiate ER membrane penetration. This reaction requires disruption of Py's disulfide bonds. Here, we found that the PDI family members ERp57, PDI, and ERp72 facilitate virus infection. However, while all three proteins disrupt Py's disulfide bonds in vitro, only ERp57 and PDI operate in concert with ERp29 to unfold the VP1 C-terminal arm. An alkylated Py cannot stimulate infection, implying a pivotal role of viral free cysteines during infection. Consistent with this, we found that although PDI and ERp72 reduce Py, ERp57 principally isomerizes the virus in vitro, a reaction that requires viral free cysteines. Our mutagenesis study subsequently identified VP1 C11 and C15 as important for infection, suggesting a role for these residues during isomerization. C11 and C15 also act together to stabilize interpentamer interactions for a subset of the virus pentamers, likely because some of these residues form interpentamer disulfide bonds. This study reveals how a PDI family functions coordinately and distinctly to promote Py infection and pinpoints a role of viral cysteines in this process.
doi:10.1128/JVI.01855-10
PMCID: PMC3067762  PMID: 21159867
4.  N-(n-Dec­yl)-4-nitro­aniline 
N-(n-Dec­yl)-4-nitro­aniline, C16H26N2O2, crystallizes with two essentially planar mol­ecules in the asymmetric unit. The decyl chains are fully extended in an anti conformation. The mol­ecules pack in planar layers, within which mol­ecules are linked into chains by two approximately linear N—H⋯O hydrogen bonds between the amine N atom and one O atom of the nitro group of an adjacent mol­ecule. These mol­ecular chains propagate via inter­leaving of the decyl chains to form the two dimensional sheets. The sheets are associated exclusively via non-bonded contacts. The structure has features in common with those of other N-alkyl-4-nitro­anilines, but also subtle differences in packing.
doi:10.1107/S1600536808003310
PMCID: PMC2960866  PMID: 21201892

Results 1-4 (4)