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Tobias Raisch

@traisch.bsky.social

160 followers 180 following 27 posts

Biochemist and Structural Biologist | Project group leader MPI Dortmund | He/him | Vegan bike punk #LeaveNoOneBehind

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Reposted by Tobias Raisch

Nature @nature.com · 18d

Nature research paper: Myeloperoxidase transforms chromatin into neutrophil extracellular traps

go.nature.com/4pxKgV6

Myeloperoxidase transforms chromatin into neutrophil extracellular traps - Nature

Myeloperoxidase, a highly expressed neutrophil protein, disassembles nucleosomes, facilitating neutrophil extracellular trap (NET) formation, and binds stably to NETs extracellularly.

Reposted by Tobias Raisch

Max Planck Institute for Infection Biology @mpiib-berlin.mpg.de · 19d

📃 New paper alert!
The Zychlinsky Lab and @raunser-lab.bsky.social identified the first protein that converts chromatin into an immune effector: Myeloperoxidase transforms chromatin into neutrophil extracellular traps. Now published in @nature.com:
www.nature.com/articles/s41...

Myeloperoxidase transforms chromatin into neutrophil extracellular traps - Nature

Myeloperoxidase, a highly expressed neutrophil protein, disassembles nucleosomes, facilitating neutrophil extracellular trap (NET) formation, and binds stably to NETs extracellularly.

Tobias Raisch @traisch.bsky.social · 20d

Thanks Weijie!

Tobias Raisch @traisch.bsky.social · 20d

Besides the really cool science, this was a fun collaboration within the @maxplanck.de between @mpiib-berlin.mpg.de and @mpi-dortmund.bsky.social.

And I guess it is only the beginning, as there are still many mysteries of NETs that @garth-burn.bsky.social, Sebastian and I want to uncover...

Tobias Raisch @traisch.bsky.social · 20d

As a final note, this whole process is highly relevant not only for killing pathogens, but also in autoimmune diseases.

We could also show that sputum of cystic fibrosis patients contains high levels of MPO functionalised NET-like structures.

Immunofluorescence images of CF patient sputum samples stained for DNA and MPO, respectively. The layover on the right shows that MPO and DNA nicely overlap.

Tobias Raisch @traisch.bsky.social · 20d

MPO monomers, a minor fraction of the native MPO pool, can also bind to nucleosomes.

But, unlike dimers, they cannot disassemble the nucleosomes and remain stably associated.

This means that most nucleosomes will be evicted during NETosis, but some are protected by MPO monomers and end up in NETs

Scheme of the NETosis mechanism and MPO's dual role in it. Panel (1) shows chromatin and MPO monomers and dimers binding to it. In panel (2) we see that both will bind to the acidic patch on nucleosomes. Step (3) is the decondensation by prevention of hitone stacking upon MPO binding. In the case of MPO dimers, there is a clash with the nucleosomall DNA. (4) nucleosomes bound by MPO dimers have mostly disassembled, so that almost only MPO monomers are still attached to mature NETs. Their role is the production of reactive oxygen species at the site of inflammation.

Tobias Raisch @traisch.bsky.social · 20d

Taken together, our data explain how MPO can achieve its dual role in NETosis (i.e. the generation of NETs) and be also a constitutent of mature NETs. The key lies in the dimerization!

MPO dimers, which are most abundant in neutrophils, will destabilized the nucleosome's DNA and lead to disassembly

Tobias Raisch @traisch.bsky.social · 20d

Finally, we went back into "real" NETs and saw by #CryoET that (1) NETs are highly complex and contain more than just decondensed chromatin, and (2) that they contain nucleosomes decorated with something that might be MPO.

On the left, we see a segmented tomogram of a NET. The tomogram is very crowded. In the center we have a massivebundle of protein filaments. It is surrounded by a tight meshwork of DNA filaments that that are decorated with protein particles. In addition, the tomogram contains also vesicles and granules.
On the right side, a cryo-ET reconstruction of a nucleosome is show featuring a prominent extra density that would be in agreement woth bound MPO. However, the resolution of the reconstruction does not allow to be certain.

Tobias Raisch @traisch.bsky.social · 20d

We see this disorder of the DNA terminus in all our reconstructions when MPO dimers are present, and only then. A strong indication that this is what actually destabilises the nucleosome!

Indeed, only MPO dimers and not monomers can lead to unwrapping of DNA, making it accessible for nucleases:

DNA deprotection assay in which a nucleosome was assembled with a DNA carrying a restriction site. Only when the DNA is unwrapped, it can be cleaved by the restriction enzyme. In the picture, we see a DNA gel in which we have lanes with one band (uncut long nucleosomeal DNA9 or two lower bands (cut DNA pieces). The left two lanes are uncut and cut DNA as marker. In the third lane, the nucleosome has been expose to MPO dimers, and the band pattern shows that all DNA is cut, meaning the DNA was unwrapped from the nucleosome. In the next five lanes, different concentrations of monomeric MPO were used, but in none the DNA was strongly cut, meaning that monomeric MPO cannot unrwap it from the nucleosome. The right two lanes are negative controls using catalase and HRP, respectively.

Tobias Raisch @traisch.bsky.social · 20d

A tiny difference in the nucleosome structures is that once the second MPO unit is present, there would be a clash with the DNA terminus.

As a consequence, in the presence of MPO dimer the DNA has to locally unwrap and becomes disordered, as visible in this morph:

Tobias Raisch @traisch.bsky.social · 20d

MPO dimers bind to the nucleosome in an identical way as the monomers we had seen before.

But the second MPO unit contacts the DNA at several positions. And this is important for understanding how it can destabilise the nucleosome.

Structure of the native MPO dimer bound to nucleosome

Tobias Raisch @traisch.bsky.social · 20d

After changing experimental conditions, we could determine intermediate structures of native MPO with nucleosomes. And we found several molecular species in these samples:

1. Free nucleosomes and MPO
2. MPO monomers bound to nucleosome
3. MPO dimers bound to nucleosome

Tobias Raisch @traisch.bsky.social · 20d

Okay, great. But what about native MPO? Does it behave similarly as the recombinant one?

Not exactly! Instead, it rapidly disassembles nucleosomes when we reconstitute both, leaving behind only MPO bound to DNA! That was a surprise, since we did not add any other factors such as ATP.

Electron micrograph from cryo-EM of nucleosomes incubated with native MPO. Almost no nucleosomes are visible, but a lot of free DNA and small proteins representing MPO and probably also histones. On the right, representative 2D classes of MPO dimers are shown with a filamentous thin density next to them, which is DNA.

Tobias Raisch @traisch.bsky.social · 20d

We then reconstituted nucleosomes with a recombinant MPO that resembles a monomeric precursor of native (dimeric) MPO.

By #CryoEM, we saw that MPO stably binds to the acidic patch of the nucleosome, an a binding mode similar to many other nucleosome interactors.

Tobias Raisch @traisch.bsky.social · 20d

The first thing we found is that MPO associates with nucleosomes in mature NETs.

This interaction is direct and requires intact nucleosomes.

Micrographs of super-resolution fluorescence microscopy (STED). Three panels represent stainig for MPO (left) and nucleosomes (middle) as well as a layover of both (right). The nucleosome channel revelas a fine meshwork representing NET filaments. MPO correlated with these filaments, but stains only discrete spots.

Tobias Raisch @traisch.bsky.social · 20d

Intriguingly, #myeloperoxidase (MPO) is a key player both for the chromatin decondensation during NETosis and it also decorates mature NETs.

It produces reactive oxygen species and is one of the most important bactericidal enzymes of neutrophils.

Cartoon view of myeloperoxidase (MPO). MPO is a homodimer, each monomer consists of a heavy and a light chain. It contains a heme cofactor.

Tobias Raisch @traisch.bsky.social · 20d

So, in essence, neutrophils transform their own chromatin into a weapon against pathogens.

But how exactly does this work? This is where we came into play.

Together with our colleagues (and now good friends) from @mpiib-berlin.mpg.de, we uncovered important steps in NETosis.

Tobias Raisch @traisch.bsky.social · 20d

During #NETosis, neutrophils decondense their #chromatin by disassembling #nucleosomes. Then, their nucleus breaks down and the cells burst open. The decondensed chomatin then forms the NET.

As #NETs are sticky and functionalised with bactericidal enzymes, they can trap and kill bacteria.

Scheme of how NETosis works. Neutrophils contain many granules that are filled with bactericidal enzymes. Once the cells are stimulated by the presence of pathogens, these granules release their content to the extracellular space, but also into the nucleus.
Inside the nucleus, MPO and other factors lead to decondensation of chromatin, i.e. eviction of nucleosomes.
The nucleus disintegrates and subsequently, the neutrophil bursts open and releases the decondensed chromatin to the extracellular space. There the NET can entrap the pathogen. As NETs are functionalized with bactericidal enzymes, they can directly kill pathogens they contact.

Tobias Raisch @traisch.bsky.social · 20d

Neutrophils are the master killers of the innate immune response. They can hunt down bacteria and eliminate them using a wide range of mechanisms, including phagocytosis and the release of ROS-generating enzymes including.

And they can form so-called Neutrophil Extracellular Traps (NETs).

Schematic drawing of a neutrophil cell and its mechanism of action. Neutrophils can leave the bloodstream to reach sites of infection/inflammation. They can uptake bacteria by phygocytosis and digest them afterwards. Also, they contain granules filles with bactericidal enzymes. Once those are released to the extracellualr space, they generate reactive oxygen species that are cytotoxic. They also release cytokines to further stimulate the immune response. And finally, they can decondense their chromatin in a process calles NETosis and release this decondensed chromatin to the extracellular space as so-called Neutrophil extracellular traps (NETs) that can entangle and kill bacteria.

Tobias Raisch @traisch.bsky.social · 20d

We just published this paper of which I am immensely proud: www.nature.com/articles/s41...

In the study, we uncover how the enzyme MPO disassembles #nucleosomes in a process called NETosis, a funky mechanism of #neutrophils to kill #pathogens that you might have never heard of.

Let's dive in... 🧵

Myeloperoxidase transforms chromatin into neutrophil extracellular traps - Nature

Myeloperoxidase, a highly expressed neutrophil protein, disassembles nucleosomes, facilitating neutrophil extracellular trap (NET) formation, and binds stably to NETs extracellularly.

Tobias Raisch @traisch.bsky.social · Jul 1

Main focus of the job will be Cryo-EM of membrane protein comolexes as well as protein/nucleic acid complexes involved in innate immunity.

For more information, check this:

www.mpi-dortmund.mpg.de/news/jobs/po...

PostDoc Position (m/f/d) in Structural Biology of Membrane Protein Complexes

www.mpi-dortmund.mpg.de

Tobias Raisch @traisch.bsky.social · Jul 1

I have an open postdoc position in my group at @mpi-dortmund.bsky.social @maxplanck.de!

If you are passionate about structural biology and/or membrane proteins, apply today!

The position is initially funded for two years, starting from this October. Deadline for application is on July 15th.

Tobias Raisch @traisch.bsky.social · Nov 21

Reminds me of something that happened a long, long time ago in Tübingen 🙈

Tobias Raisch @traisch.bsky.social · Nov 4

Yes, indeed. Thank you!

Tobias Raisch @traisch.bsky.social · Nov 4

Thanks Eugene :)