Publications by authors named "Colleen Hui"

5 Publications

  • Page 1 of 1

Single-cell visualization and quantification of trace metals in lysosome-related organelles.

Proc Natl Acad Sci U S A 2021 Apr;118(16)

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.
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http://dx.doi.org/10.1073/pnas.2026811118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072209PMC
April 2021

Ligand-induced allostery in the interaction of the heme binding protein with heme oxygenase.

Proc Natl Acad Sci U S A 2017 03 13;114(13):3421-3426. Epub 2017 Mar 13.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201;

A heme-dependent conformational rearrangement of the C-terminal domain of heme binding protein (PhuS) is required for interaction with the iron-regulated heme oxygenase (HemO). Herein, we further investigate the underlying mechanism of this conformational rearrangement and its implications for heme transfer via site-directed mutagenesis, resonance Raman (RR), hydrogen-deuterium exchange MS (HDX-MS) methods, and molecular dynamics (MD). HDX-MS revealed that the apo-PhuS C-terminal α6/α7/α8-helices are largely unstructured, whereas the apo-PhuS H212R variant showed an increase in structure within these regions. The increased rate of heme association with apo-PhuS H212R compared with the WT and lack of a detectable five-coordinate high-spin (5cHS) heme intermediate are consistent with a more folded and less dynamic C-terminal domain. HDX-MS and MD of holo-PhuS indicate an overall reduction in molecular flexibility throughout the protein, with significant structural rearrangement and protection of the heme binding pocket. We observed slow cooperative unfolding/folding events within the C-terminal helices of holo-PhuS and the N-terminal α1/α2-helices that are dampened or eliminated in the holo-PhuS H212R variant. Chemical cross-linking and MALDI-TOF MS mapped these same regions to the PhuS:HemO protein-protein interface. We previously proposed that the protein-protein interaction induces conformational rearrangement, promoting a ligand switch from His-209 to His-212 and triggering heme release to HemO. The reduced conformational freedom of holo-PhuS H212R combined with the increase in entropy and decrease in heme transfer on interaction with HemO further support this model. This study provides significant insight into the role of protein dynamics in heme binding and release in bacterial heme transport proteins.
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http://dx.doi.org/10.1073/pnas.1606931114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380046PMC
March 2017

Quantitating PrP Polymorphisms Present in Prions from Heterozygous Scrapie-Infected Sheep.

Anal Chem 2017 01 21;89(1):854-861. Epub 2016 Dec 21.

Veterinary Faculty, Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes (CIEETE), Universidad de Zaragoza , 50013, Zaragoza Spain.

Scrapie is a prion (PrP) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep's normal cellular prion protein (PrP). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [MLGSXMSRPL (X = A or V), YXENMY (X,= H or R), and YRPVDXY (X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrP or PrP. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrP isolated from sheep heterozygous for their PrP proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrP. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrP and samples containing only the PK-resistant PrP. These results show that heterozygous animals contain PrP that is composed of significant amounts of both PrP polymorphisms.
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http://dx.doi.org/10.1021/acs.analchem.6b03822DOI Listing
January 2017

Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.

Anal Chem 2014 May 2;86(10):4698-706. Epub 2014 May 2.

Western Regional Research Center, United States Department of Agriculture , Albany, California 94710, United States.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.
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http://dx.doi.org/10.1021/ac402930rDOI Listing
May 2014

Oxidation of methionine 216 in sheep and elk prion protein is highly dependent upon the amino acid at position 218 but is not important for prion propagation.

Biochemistry 2013 Mar 15;52(12):2139-47. Epub 2013 Mar 15.

Western Regional Research Center, United States Department of Agriculture , Albany, California 94710, United States.

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GENFTETDIK (sheep and elk) and ESQAYYQR (sheep) or ESEAYYQR (elk). This approach required much smaller amounts of tissue (600 μg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation.
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http://dx.doi.org/10.1021/bi3016795DOI Listing
March 2013