Protein Power: Distortion of the Lipid Bilayer by Rhomboid Proteases

Paper: Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion, by Alex J. B. Kreutzberger, Ming Ji, Jesse Aaron, Ljubica Mihaljević, and Siniša Urban https://www.ncbi.nlm.nih.gov/pubmed/30705155

The traditional paradigm for stable,
yet dynamic cellular membranes relies on the stability of phospholipids and
their strength in numbers. Proteins are imagined merely as working members of
this system, either residing on the surface of the membrane or having a hydrophobic
transmembrane region capped with two hydrophilic functional caps. In recent
years, researchers have discovered a new class of protease which seem to push
the limits of this model. Rhomboid proteins, first discovered in Drosophila
melanogaster but later found to be ubiquitous in all life¹, are
a class of intermembrane serine protease. Their function in the cell is to
cleave small peptides off other intermembrane proteins, which is the initiating
and irreversible step in certain signaling pathways. They use a serine-histidine
dyad for catalysis, unlike the Ser-His-Asp triad common in many other enzymes. The
human genome encodes five rhomboids, four residing in the plasma membrane,
called rhomboid-like 1-4 (RHBDL1-4), and one in mitochondria².

Kreutzberger et al. investigated the unusual structure of rhomboids and their method of diffusion into the membrane³.  They were previously known to contain six transmembrane α-helices, like normal transmembrane proteins, but they also have an α-helical hairpin protruding out from the core, perpendicular to the other helices¹. It is the effect of this and other unique structures that Kreutzberger et al. found distort the membrane in a unique way (Figure 1). Rhomboids have previously been linked to diseases as diverse as Parkinson’s and carcinogenesis³. Thus, it is worthwhile to research rhomboids to further deduce their functions. The biological workaround found by the researchers may be exploited by other proteins, which is important to know because of the possibility of manipulating this property biochemically or biophysically.

Clues leading up to the researchers’ discovery were dots for them to eventually connect. Sanders and Hutchinson found that, when interacting, rhomboids distorted some annular lipids. They speculated that, in membranes, rhomboids distort lipids to make room for cleavage, which occurs in the nonpolar membrane, not exposed to the cytosol. They also mentioned rhomboids could make the adjacent bilayer thinner but didn’t go into detail because they were studying something else⁴. Bondar et al. ran simulations of membranes that showed nonuniform thinning of the lipids in the presence of rhomboids and their odd protrusions⁵.

Ramadurai et al. looked at diffusion kinetics of several membrane proteins. They used the Saffman-Delbrück model, which plots the rates of diffusion in and through the plasma membrane. In general, smaller proteins diffuse faster, and this trend makes a smooth curve of molecular radius plotted against diffusion rate. The only deviants from the Saffman-Delbrück model were the two rhomboids they analyzed, and they deviated by a lot. This strongly suggests rhomboids utilize an alternative diffusion pathway⁶.

Within this growing context of aberrant diffusion, Kreutzberger et al. ran several experiments on three distinct model systems. Cell lines from the human embryonic kidney (HEK239T) and Drosophila (S2R+) were used as biological standards, the former for the possibility of medical applications and the latter because, to date, rhomboids have been studied to the greatest detail in the fruit flies. The third system was a synthetic, in vitro lipid bilayer, used for the more quantitative experiments and compared to observations in vivo.

In all rhomboid experiments, which used RHBDL2, the rhomboid proteases were labeled with HaloTag, which came from a recombinant plasmid in the in vivo systems. This 297-residue peptide was attached to the rhomboid using CRISPR-Cas9 and detected in various ways depending on the experiment. In one experiment, they tethered HaloTag to quantum dots to selectively observe rhomboid diffusion. To make sure the cytoskeleton was not playing a part in rhomboid diffusion, they ran tests with microtubule destabilizing or actin depolymerizing drugs, both with no effect on diffusion rate. To test if diffusion into the membrane was the rate-limiting step in membrane protein cleavage, they used ionomycin precomplexed with magnesium to lower the transition temperature of phospholipids, essentially making the membrane more fluid and increasing diffusion rate. This consequently increased the rate of cleavage, producing kinetic evidence that diffusion is indeed the rate-limiting step in rhomboid cleavage. This is important because it means there could be a large evolutionary pressure for faster diffusion by rhomboid proteases.

The large cytosolic appendage attached to RHBDL2’s N-terminus was speculated to aid with diffusion. In experiments where the cytosolic domain was removed, diffusion rates dropped by 30%, indicating this is the case. Follow-up experiments showed that the appendage repositions the rhomboid in the membrane, further enhancing its ability to distort lipids. Likewise in the in vitro bilayer, replications of Ramadurai’s research were performed. They again found that rhomboids broke the Saffman-Delbrück limit, and even diffused faster than some relatively small peptides.

The most important experiment, however, was the varying of membrane thickness. Rhomboid’s belt, or its intermembrane α-helix region, is thinner than the typical membrane. When in vitro membranes were made thinner by using shorter fatty acid chains, they fit the size of RHBDL2’s belt. Now, since there was no hydrophobic mismatch, the reverse of the previous diffusion rate trend was observed, with normal proteins diffusing faster than rhomboids. This is strong evidence that the advantage rhomboids have is their hydrophobic mismatch with typical membranes, which causes them to be less confined by phospholipids and hence more mobile. This also explains how rhomboids avoid the Saffman-Delbrück limit, because they don’t even fit the conditions of the model in living cells. Other experiments were conducted, but they either assisted the preceding experiments or contributed marginally to the conclusion.

The authors left little doubt that hydrophobic mismatch was the culprit behind the rhomboids’ efficient diffusion in membranes. This inference leaves open the possibility of the natural next step, follow-up research to find other proteins that use the same mechanism for different functions. It could also someday explain the function of other rhomboids which have lost their catalytic ability but remain in cells. These proteins’ ability to distort membranes in a way unlike any other could be used as a mediating factor in other membrane proteins’ function⁷. In a broader sense, studies like Kreutzberger’s inspire other to think outside the box (or more accurately, to distort the box). Permanently challenging the canonical view of transmembrane proteins may be the most lasting effect of this paper.

Works Cited

1. Kinch, L. & Grishin, N. Biochim. Biophys. Acta 1828, 2937-2943 (2013). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505743/
2. Urban, S. Cell 167, 1898 (2016). https://www.sciencedirect.com/science/article/pii/S0092867416316531?via%3Dihub
3. Kreutzberger, A. et al. Science 363, 1-9 (2019). https://www.ncbi.nlm.nih.gov/pubmed/30705155
4. Sanders, C. & Hutchinson, J. Curr. Opin. Struct. Bio. 51, 80-91 (2018). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158105/
5. Bondar, A. et al. Structure 17, 395-405 (2009). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158105/
6. Ramadurai, S. et al. Biophys. J. 99, 1482-1489 (2010). https://www.ncbi.nlm.nih.gov/pubmed/20816060
7. Freeman, M. Annu. Rev. Cell Dev. Biol. 80, 235-254 (2014). https://www.ncbi.nlm.nih.gov/pubmed/25062361

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