Thorpe Lab

oxidative protein folding

QSOX as a “bad actor” in (some forms of) breast cancer

The "bad actor" quote comes from a recent paper by Douglas Lake and collaborators: "Expression of Quiescin Sulfhydryl Oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in luminal B breast cancer"

The enzyme: readers of earlier blogs may know that Quiescin-sulfhydryl oxidases (QSOXs) catalyze disulfide bond generation within conformationally flexible peptides and proteins. In the enzymological equivalent of a "bucket brigade", pairs of electrons are passed from the dithiol-containing substrate to molecular oxygen as shown in the Figure. In the crystal structure of the mouse QSOX1 enzyme the deep blue [Trx1] domain is handing over a pair of electrons to the green [ERV] domain prior to the final stages in catalysis (steps 3 and 4).

QSOX Blog breast c

 

Breast cancer: According to the American Cancer Society, women have a 1 in 8 lifetime probability of developing breast cancer – accounting for 30% of new cancer cases among women in 2012. There are multiple categories of breast cancer: the one that Lake and colleagues were studying was luminal B breast cancer – an estrogen receptor-positive subtype.

The first paper: Lake and colleagues found that QSOX1 protein is highly over-expressed in luminal B cancers and cell lines. Knockdowns of QSOX1 protein levels suppressed cell proliferation, and dramatically reduced the invasive potential of these tumor cells. Importantly, exogenously added QSOX1 enzyme restored the invasive potential of QSOX-depleted cells. Lake and coworkers suggest that elevated QSOX levels lead to increased activity of matrix metalloproteinase 9 with a consequent stimulation of degradation of ECM components and an increased propensity for metastasis.
Lake and coworkers used the GOBO database "Gene Expression Based Outcome for Breast Cancer Online" to make the case that elevated QSOX1 message levels are associated with a poor prognosis in both luminal A and B cancers.

In a second study " Elevated Transcription of the Gene QSOX1 Encoding Quiescin Q6 Sulfhydryl Oxidase 1 in Breast Cancer", Soloviev et al. analyzed expressed sequence tag (EST) and serial analysis of gene expression (SAGE) databases. They also concluded that QSOX1 is highly overexpressed in some breast cancers. They used quantitative PCR to analyze a series of breast tumors, and observed a strong correlation between QSOX1 expression levels and the progression of the disease.

Other cancers: Previous posts have reported that overexpression of QSOX1 correlates with the invasive potential of prostate and pancreatic cancer tumors. In addition to modulating the activity of MMP9, what other activities of QSOX1 contribute to the increased fitness of particular tumor cells? Are these effects confined to extracellular disulfide bond regeneration/remodeling – or is the other product of QSOX catalysis - hydrogen peroxide - a factor?


But is QSOX really a "bad actor"?  In a paper published in 2012, Pernodet et al. reached exactly the opposite conclusion to the studies reported above. They analyzed 217 invasive ductal carcinoma patients in their paper "High expression of QSOX1 reduces tumorogenesis, and is associated with a better outcome for breast cancer patients" Pernodet et al. found an inverse correlation between QSOX expression levels and the aggressiveness of the breast tumors they examined.

They report that "QSOX1 also reduces the invasive potential of cells by reducing cell migration and decreases the activity of the matrix metalloproteinase, MMP-2, involved in these mechanisms. Moreover, in vivo experiments show that QSOX1 drastically reduces the tumor development"

This chemist-contributor is now in a muddle! How to reconcile the differences between this work and the two studies reported at the beginning of this post – do they reflect differences in tumor populations studied? Hopefully the potential importance of these observations will encourage a more searching and comprehensive examination of the role of QSOX in tumorigenesis.

May 19th addendum to original May 8, 2013 post: for a more detailed comparison of the papers, see a recent Editorial in Breast Cancer Research by Postovit and colleagues "Illuminating luminal B: QSOX1 as a subtype-specific biomarker" [PubMed or Publisher]

Conduct Unbecoming

We have published a body of work on a small sulfhydryl oxidase – reasonably fundamental stuff I thought.  We found new substrates, new enzymatic activities, explored enzymatic mechanisms and, with colleagues, solved crystal structures.  So I was disconcerted that a speaker at a recent conference never mentioned any of our contributions in his talk on this same enzyme (I was there, and we have other witnesses!).   There was no acknowledgment in the introduction, nothing in the middle and nothing at the end.

So what is a conferee to do?  Should I have complained in the question-and-answer period and sounded like a whiner?  Should I raise this with the speaker privately and listen to his excuses? Should I send an aggrieved letter?  I did none of these things – maybe the speaker meant to credit our work but nerves overwhelmed him.  Or maybe he just forgot.  Give the fellow the benefit of the doubt.

My annoyance was rekindled recently when I read one of his papers and found that our contributions to the same enzyme were again essentially unacknowledged – an uninformed reader would conclude that the whole intellectual framework of the paper originated with the authors.  Yet many of the central ideas had been clearly identified years earlier.  Now we don’t need witnesses - the literature is our record!

This sort of behavior is, at best, ungracious.  What does he lose by citing the literature appropriately and by acknowledging in talks that others have played a role in the development of the field?  A failure to cite relevant work is also unethical – to quote from the Ethical Guidelines for Publication from the ACS:

“... An author is obligated to perform a literature search to find, and then cite, the original publications that describe closely related work….”

“Colin, just get over it – these things happen all the time” …. I was given this sage advice almost 20 years ago (for a different case entirely!).  The very next day my “turn-the-other–cheek” colleague heard his own work characterized with insufficient deference.  This time it was personal, and he went ballistic!

Maybe that’s what I’ll do next time!

Expanding the Biological NMR Toolbox

What’s your favorite element?  - for many scientists studying oxidative protein folding it would have to be sulfur.  Not only does the oxidation of cysteinyl sulfur generate the constellation of structural disulfide bonds found in secreted proteins, but cysteine residues are an essential catalytic ingredient in the enzymes that form and isomerize these very disulfide linkages.  While carbon, nitrogen and hydrogen can be routinely probed by nuclear magnetic resonance, the application of biological NMR to sulfur is impractical - there is no suitable sensitive NMR-active isotope for this critical player in oxidative protein folding.

Now Sharon Rozovsky and her colleagues have devised a simple way to substitute the NMR-active 77Se isotope for sulfur in recombinant proteins [PubMed].  Escherichia coli cells are grown in minimal media until they approach sulfur starvation, and then the medium is supplemented with ratios of Se (as selenite) to S (as sulfate) that are needed to achieve the desired incorporation ratio of Se/S.  Anyone who expresses recombinant proteins in minimal media should be able to make this method work following the protocol outlined in Schaefer et al. [PubMed].

One of the proteins they have studied is augmenter of liver regeneration (ALR) – an enzyme that drives disulfide bond generation in the intermembrane space of the mitochondrion.  The short form of ALR is a covalent homodimer (32 kDa) containing four structural disulfide bonds, two catalytic disulfides, and two FAD prosthetic groups.  Using the random selenium incorporation method of Rozovsky and coworkers, Se/S ratios of up to 90% are readily achievable for human ALR. The resulting Se-ALR enzyme is almost as stable as the wild type protein, and the substituted protein retains significant enzymatic activity.  Importantly, multiple selenocysteine and selenomethionine resonances can now be visualized by conventional solution NMR without the need for high field strengths or lengthy acquisition times.

But what are the structural consequences for such a wholesale substitution of sulfur for selenium in ALR?  Schaefer et al. were able to crystallize the 90% substituted protein and compare it to the coordinates of the human ALR protein already solved in the laboratory of Brian Bahnson [PubMed].  On the left you can see the structure of Se-ALR refined to a resolution of 1.5 Angstroms with an occupancy of 90% Se / 10% S [PDB link] .  The protein fold is essentially identical to that of native human ALR.

alr_selenium_new

 The right panels show the reduction of the enzyme by the model substrate dithiothreitol (DTT) monitored through the changes in the flavin spectrum.  (Top panel is wild-type ALR; bottom panel is Se-rich ALR.  Note the new long-wavelength absorbance feature that intervenes before the accumulation of the blue semiquinone).

In sum, the work of Rozovsky and coworkers should help to put selenium into the biological NMR toolbox - as an environmental probe, and for the characterization of redox and ligation states. While attention has been drawn to the differences in reactivity between sulfur and selenium – they really do share lots in common.

So if you want to try an NMR-active surrogate for sulfur in your favorite protein … give 77Se a spin!

Notes

1)   The paper: Stephanie A. Schaefer, Ming Dong, Renee P. Rubenstein, Wayne A. Wilkie, Brian J. Bahnson, Colin Thorpe and Sharon Rozovsky. 77Se Enrichment of Proteins Expands the Biological NMR Toolbox. Journal of Molecular Biology (2012 [PubMed].

2)  Sharon Rozovsky's website is here

3)  Of course genetic incorporation of Se provides for site specificity - but to do this wholesale for a multicysteine-containing protein would generally require an unacceptable number of mutations to generate SECIS elements for each position.

4)  In terms of “disulfides” 90% selenium incorporation corresponds to  1% -S-S-, 18% -S-Se- (or -Se-S-) and 81% -Se-Se-.  So the majority of crosslinks in the selenium-ALR structure depicted in the Figure represent diselenides.

5)  The PDB coordinates for the Selenium-rich form of ALR are to be found here

 

QSOX and the heart

Shortness of breath (a.k.a. dyspn(o)ea) is caused by a wide range of conditions including acute anxiety, asthma, several lung diseases and infections, and various forms of heart failure.  In a recent paper, Mebazaa et al. [PubMed] sought new serum markers that would specifically identify those patients whose labored breathing was due to heart failure.

qsox_and_your_heart_new

 

The authors screened proteins in blood serum using an unbiased mass-spectrometric proteomics approach.  Encouragingly, they identified the B-type natriuretic peptide (BNP) - a widely-used marker for the assessment of heart failure.  However they also found that the sulfhydryl oxidase, QSOX1, was a potentially useful diagnostic biomarker for heart failure.  They state:   “ … we found a novel biomarker QSOX1 to be highly sensitive and specific for … diagnosis in patients with acute dyspnoea with a performance equaling the gold standard biomarkers BNP and NT-proBNP. We further demonstrate that the combination of QSOX1 and BNP markedly reduces false positives and exerts the best specificity for … diagnosis in patients with dyspnoea.”

They also write “We found that QSOX1 is unaffected by many of the factors that weaken the value of BNP, and the combination of BNP plus QSOX1 provided the best sensitivity and specificity for ADHF in patients with dyspnoea.”

So why are elevated serum levels of QSOX1 associated with heart disease? Good question!  The authors speculate that there might be a connection QSOX and the need to insert a disulfide bond in the BNP peptide.  However, in our opinion, the vigorous disulfide bond-generating activity of QSOX would likely make short work of the < nM levels of these BNP peptides that are found in sera.  Perhaps enhanced QSOX1 secretion accompanies the remodeling of the extracellular matrix associated with damage to heart muscle.  Whatever the explanation, the results of Mebazza et al. suggest that QSOX has the potential to be a useful diagnostic indicator for heart failure.

Addenda

Although not mentioned in Mebazaa et al., or in an associated commentary, the connection between the human growth factor Quiescin Q6 and the disulfide bond-forming enzyme, now known as QSOX, was made by Hoober et al. and by Benayoun et al.  The discovery of QSOX on this side of the pond is recounted in an earlier article in this series.

For a description of the use of QSOX1 as a potential biomarker for pancreatic cancer see this link, and for its overexpression in prostate cancer see this link.

We note that QSOX was originally described in blood serum by Nakao and coworkers.

Frequently, a failing left ventricle is signaled by pulmonary edema and shortness of breath.  For a schematic of the pumping heart see.

 

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