Connor Rosen Profile picture
Apr 19, 2019 14 tweets 5 min read Read on X
Here’s a bit of a dive into some interesting recent work on #PhaseSeparation in cells - focusing on the role of ATP as a protein stabilizer in cells, independent of its energy-providing function. It all starts with a paper in @ScienceMagazine from 2017, science.sciencemag.org/content/356/63….
This Science paper showed that in vitro, ATP had hydrotope activity - the ability to solubilize proteins, and dissolve protein aggregates or liquid phase droplets. This function occurred at millimolar concentrations of ATP, near normal cellular levels.
There was some pretty quick push-back against the interpretation about any cellular role - pubpeer.com/publications/8…. One major point of criticism is that even at 5 mM ATP, there’s only about 1 ATP molecule per protein in a cell. So it’s hard to imagine proteome-scale activity.
Still, the paper has picked up over a hundred citations - scholar.google.com/scholar?cites=…. Most of them, however, are “drive-by” citations that don’t actually confirm the findings but simply cite hydrotope activity as a role of ATP.
Those that are directly assaying ATP activity are somewhat split - plenty of individual examples in vitro or in cells of no role for ATP in stabilization, but also some suggesting, but not fully proving, a hydrotope role in cells.
So far, I think it’s pretty tough to say this is a really well-established fact that can be applied to cellular homeostasis. One big hurdle is disassociating ATP activity as a hydrotope vs as an energy source for chaperones.
And so stories like cdn.elifesciences.org/articles/35224… and sciencedirect.com/science/articl… end up arguing for multiple roles of ATP, including chaperone-dependent roles in protein stabilization in cells. And it's not really clear how to reconcile them with a hydrotope model.
That’s why I really like this new paper in @NatureComms from @savitski_lab (nature.com/articles/s4146…). They use some really good proteome-wide mass spec techniques to profile both thermal stability and solubility in response to ATP.
The basic principle is to measure soluble protein abundance in cell lysates by mass-spec at a range of temperatures (or detergent solubilizing agents) and ATP concentrations, allowing building of melting-temp and solubility curves in response to ATP.
The experiments seem very well set up and controlled, and are pretty strongly suggestive of an energy-independent role for ATP stabilization in cells. Importantly, it’s not for all proteins, which gets around the concentration issues discussed on pubpeer.
Many proteins that are stabilized are ATP-binding proteins, and ligand binding is known to (in general) stabilize proteins, so that’s not exactly a pure “hydrotope” role. It is certainly an energy-independent role, with large effects on the proteome, but not a hydrotope.
But I think there’s sufficient evidence that a substantial number of non-ATP-binding proteins (especially IDPs and highly charged proteins, known to be prone to phase separation) are stabilized and solubilized in a manner that is more consistent with a hydrotope activity.
There’s a lot more data in the paper, certainly worth careful reading. It seems like a really solid paper and I think it really argues for a broad, but not universal, role of ATP as a biological hydrotope that previous work hadn’t fully established.
As a quick note, it was also posted early as a #preprint (biorxiv.org/content/10.110…). @NatureComms also posts peer review comments, and the feedback was very positive. This is a good example of two trends in publishing I like, preprints and open peer review!

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More from @connor_rosen

Sep 25, 2020
Here’s a thread on anti-interferon autoantibodies, viral infections, and human immunology. This is less a covid thread, and more an anti-covid thread, if anything…
Summary: anti-IFN auto-Abs may be reflective of chronic inflammation, and may pre-exist in vulnerable groups at high levels. Presence in severe covid cases may therefore reflect basal immune variation which impacts covid, rather than a special covid-specific phenomenon.
This is prompted based on the new Science paper on autoantibodies against Type I IFNs in severe covid patients (science.sciencemag.org/content/early/…). Basically, ~10% of severe covid patients had high titer anti-IFNa antibodies that were functionally neutralizing.
Read 23 tweets
Jun 2, 2020
The message is very simple. #BlackLivesMatter
I'm grateful that others have put together resources like bit.ly/ANTIRACISMRESO…, which has helped me learn to be better. So You Want To Talk About Race by @IjeomaOluo and How To Be An Antiracist by @DrIbram have been amazing, and I'm looking forward to reading deeper.
I haven't seen a similar central resource for donations, but various recommendations led me to eji.org (Equal Justice initiative, focused on criminal justice reform and education), voterparticipation.org (get people registered to vote)...
Read 5 tweets
Apr 21, 2020
The story about mitochondria being ~10° hotter than the rest of the cell, published @PLOSBiology a couple years ago (journals.plos.org/plosbiology/ar…) got some cool, albeit indirect, support recently @naturemethods#Biophysics #CellBiology
The original paper, which was mainly based on the temperature-dependent fluorescence of a mitochondria-targeting probe, was accompanied by a “Primer” (journals.plos.org/plosbiology/ar…) highlighting potential flaws and implications, a special sort-of-peer-review step by PLOS Biology.
This new awesome resource @naturemethods - nature.com/articles/s4159… - offers some intriguing orthogonal validation. This is a proteome-wide study of protein thermal stability across 13 organisms, conducted using a mass-spec-based approach.
Read 11 tweets
Nov 22, 2019
Super neat story on how cellular quality control impacts the mutational landscape of proteins - beneficial mutations in DHFR during deep mutational scanning are totally altered dependent on cellular QC #Biophysics #Evolution biorxiv.org/content/10.110… Way to go! @KortemmeLab
In an initial DMS experiment on DHFR, there were a large number (25% of all sequences!) of advantageous mutations spread across the whole protein. Reintroduction of QC protein Lon reduced the number of advantageous mutants and lowered average benefit of those mutations
Changes in selection coefficient (the “advantageous-ness”) were most striking at hydrophobic/aromatic residues and buried residues - and these correlate with Tm changes of variants. So Lon seems to be imposing higher standards on DHFR, particularly for destabilizing core mutants
Read 5 tweets
Nov 20, 2019
A couple interesting bits of preliminary data on Langerhans cells and the huLangerin mouse used to study them. Effects of developmental absence of LCs on keratinocytes and T cells, and huLangerin-YFP labels some neurons (check your Cre mice!) #Genetics #Immunology
First - effect of loss of LCs in the huLangerin-DTA mice - biorxiv.org/content/10.110…. Bulk RNA-seq showed changes in keratinocytes and dendritic epidermal T cells, including cell-type specific changes (e.g. loss of IL17 pathway in DETCs).
Unfortunately, underlying data (either gene expression tables or raw RNA-seq data) don’t currently seem available, but hopefully the authors get that up shortly. Will be interesting to look at and prompt some hypotheses about how LCs control homeostasis and development.
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Nov 18, 2019
CYTOF analysis of human neutrophils - 7 populations with differing phagocytosis, ROS, and FACS-compatible surface marker phenotypes. Changes in distribution between healthy + melanoma patients. #Immunology #Cancer #Neutrophils biorxiv.org/content/10.110…
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Interesting to note the phagocytosis-SSC staining in Fig4A - big changes in SSC for some populations with zymosan, others not so much - degranulation, different phagosomes…? Similarly, bimodal peaks for ROS production in same populations… Image
Read 4 tweets

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