Tag Archives: innate immune system

RIPK1

The innate immune system is intrinsically fascinating, dealing with invaders long before antibodies or cytotoxic cells are on the scene.  It is even more fascinating to a chemist because it works in part by forming amyloid inside the cell.  And you thought amyloid was bad.

The system becomes even more fascinating because blocking one part of it (RIPK1) may be a way to treat a variety of neurologic diseases (ALS, MS,Alzheimer’s, Parkinsonism) whose treatment could be improved to put it mildly.

One way to deal with an invader which has made it inside the cell, is for the cell to purposely die.  More and more it appears that many forms of cell death are elaborately programmed (like taking down a stage set).

Necroptosis is one such, distinct from the better known and studied apoptosis.   It is programmed and occurs when a cytokine such as tumor necrosis factor binds to its receptor, or when an invader binds to members of the innate immune system (TLR3, TLR4).

The system is insanely complicated.  Here is a taste from a superb review — unfortunately probably behind a paywall — https://www.pnas.org/content/116/20/9714 — PNAS vol. 116 pp. 9714 – 9722 ’19.

“RIPK1 is a multidomain protein comprising an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The intermediate domain of RIPK1 contains an RHIM [receptor interacting protein (rip) homotypic interaction motif] domain which is important for interacting with other RHIM-containing proteins such as RIPK3, TRIF, and ZBP1. The C-terminal DD mediates its recruitment by interacting with other DD-containing proteins, such as TNFR1 and FADD, and its homodimerization to promote the activation of the N-terminal kinase domain. In the case of TNF-α signaling, ligand-induced TNFR1 trimerization leads to the assembly of a large receptor-bound signaling complex, termed Complex I, which includes multiple adaptors (TRADD, TRAF2, and RIPK1), and E3 ubiquitin ligases (cIAP1/2, LUBAC complex).”

Got that?  Here’s a bit more

“RIPK1 is regulated by multiple posttranslational modifications, but one of the most critical regulatory mechanisms is via ubiquitination. The E3 ubiquitin ligases cIAP1/2 are recruited into Complex I with the help of TRAF2 to mediate RIPK1 K63 ubiquitination. K63 ubiquitination of RIPK1 by cIAP1/2 promotes the recruitment and activation of TAK1 kinase through the polyubiquitin binding adaptors TAB2/TAB3. K63 ubiquitination also facilitates the recruitment of the LUBAC complex, which in turn performs M1- type ubiquitination of RIPK1 and TNFR1. M1 ubiquitination of Complex I is important for the recruitment of the trimeric IκB kinase complex (IKK) through a polyubuiquitin-binding adaptor subunit IKKγ/NEMO . The activation of RIPK1 is inhibited by direct phosphorylation by TAK1, IKKα/β, MK2, and TBK1. cIAP1 was also found to mediate K48 ubiquitination of RIPK1, inhibiting its catalytic activity and promoting degradation.”

So why should you plow through all this?  Because inhibiting RIPK1 reduces oxygen/glucose deprivation induced cell death in neurons, and reduced infarct size in experimental middle cerebral artery occlusion.

RIPK1 is elevated in MS brain, and inhibition of it helps an animal model (EAE).  Mutations in optineurin, and TBK1 leading to familial ALS promote the onset of RIPK1 necroptosis

Inflammation is seen in a variety of neurologic diseases (Alzheimer’s, MS) and RIPK1 is elevated in them.

Inhibitors of RIPK1 are available and do get into the brain.  As of now two RIPK1 inhibitors have made it through phase I human safety trials.

So it’s time to try RIPK1 inhibitors in these diseases.  It is an entirely new approach to them.  Even if it works only in one disease it would be worth it.

Now a dose of cynicism.  Diseased cells have to die one way or another.  RIPK1 may help this along, but it tells us nothing about what caused RIPK1 to become activated.  It may be a biomarker of a diseased cell.  The animal models are suggestive (as they always are) but few of them have panned out when applied to man.

 

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You are alive because the lipid bilayer of your plasma membrane is asymmetric

You are an organism with trillions of cells. A mosquito bit you depositing millions of viruses in your tissues. The virus can reproduce only within one of your cells and it has exploited all sorts of protein protein chemistry to get in. Antibodies (if you are fortunate enough to have them) can get rid of the extracellular critters. However, 500,000 have made into the same number of your cells, and are merrily trying to reproduce.

How does the asymmetry of the lipid bilayer of your plasma membrane help you survive. If each virus infected cell killed itself before the virus reproduced, you’d survive. Although 500,000 is a large number is is less than 1 millionth of your cell total.

Well you do have intracellular defenses against viruses, called the innate immune system. One of them is a protein with the ugly name of gasdermin D. The activated innate immune system (in the form of inflammatory caspases) cleaves gasdermin. This breaks up the inhibition of the amino terminal part of gasdermin by the carboxy terminal part giving a fragment which binds to one particular membrane component (phosphatidyl serine) which makes up 20% of the inner leaflet of the cell membrane. It then forms a large diameter (to a cell 140 Angstroms is quite large) pore in the cell membrane. No cell can survive this, so it dies, releasing cellular contents (probably some viral components but not fully formed one). For details see [ Nature vol. 535 pp 111 – 116, 153 – 158 ’16 ]

Wait a minute. The toxic gasdermin fragment is also released. So how come it doesn’t kill everything in sight? Because our cellular membranes keep phosphatidyl serine confined to the inner membrane, normal cells don’t show it on their exterior, so they can be bathed in gasdermin with no ill effect. What is responsible for this asymmetry — believe it or not an ATP consuming enzyme called flippase (about this more later) which takes any phosphatidyl serine it finds on the outer leaflet and schleps it back inside the cell.

There is all sorts of elegant chemistry which explains just how gasdermin binds to phosphatidyl serine and none of the many other phospholipids found on the inner leaflet. There is more elegant chemistry explaining how flippase works (see later).

What chemistry cannot explain, is why organisms would ‘want’ an asymmetric membrane. As soon as you get into the function of a particular compound in an organism, chemistry is powerless to tell you why. Nothing else can explain how a given molecule does what it does on the molecular level but that is not enough for a satisfying explanation.

One further explanation before some hard core cellular biochemistry follows (after ***). Our cells are dying all the time. The lining of your gut is replaced every 5 days. Even the longest lasting element of your blood is gone after half a year, and most other elements are turned over at least once a month. When these cells die, they must be cleaned up, without undue fuss (such as inflammation). The cleaners are cells called macrophages. A dying cell releases chemical signals, actually called ‘eat me’, one of which is phosphatidyl serine found on the membrane fragments of a dead cell. The fact that flippases keep it on the inner leaflet means that macrophages won’t attack a normal cell.

Slick isn’t it?

***

Flippase is a MgATPdependent aminophospholipid translocase. It localizes phosphatidylserine and phosphatidylethanolamine to the inner membrane leaflet by rapidly translocating them from the outer to the inner leaflet against an electrochemical gradient. The stoichiometry between amino phospholipid translocation and ATP hydrolysis is close to one (how will the cell have enough ATP to do anything else?). The flippase is inhibited by high calcium, and by pseudosubstrates such as vanadate, acetylphosphate and para-nitrophenyl phosphate, and by SH reactive reagents such as N-ethylmaleimide and pyridyldithioethylamine (PDA) a specific inhibitor of phospholipid translocation

[ Proc. Natl. Acad. Sci. vol. 109 pp. 1449 – 1454 ’12 ] P4-ATPases are a subfamily of P-type ATPases. They transport aminophospholipids from the exoplasmic to the cytoplasmic leaflet (and are known as flippases). Man has 14 P4-ATPases, expressed in various cell types. They are thought to be similar to the catalytic subunits of the Ca++ ATPase, and the Na, K ATPase, consisting of cytoplasmic, N, P and A domains and a membrane domain made of 10 transmembrane helices (M1 – M10).

[ Proc. Natl. Acad. Sci. vol. 111 pp. E1334 – E1343 ’14 ] The P4-ATPases are thought to resemble the classic P-type ATPase cation pumps — a transmembrane domain of 10 helices and 3 cytoplasmic domains (P for phosphorylation, N for nucleotide binding and A for actuator). ATP8A2 forms an intermediate phosphorylated on aspartic acid (E2P)and undergoes a catalytic cycle similar to the sodium pump (Na+, K+ ATPase). Dephosphorylation of E2P is activated by the transported substrates phosphatidyl serine (PS) and phosphatidyl ethanolamine (PE), similar to the K+ activation of dephosphorylation in the sodium pump.

PE and PS are 10x as large as the cations transported by the sodium pump. This is known as the giant substrate problem. This work shows that isoleucine #364 (mutated in — patients with the ataxia, retardation and dysequilibrium syndrome Eur. J. Hum. Genet. vol. 21 pp. 281 – 285 ’13 aka CAMRQ syndrome ) forms a hydrophobic gate separating the entry and exit sites of PS. I364 likely directs the sequential formation and annihilation of water filled cavities (as shown by molecular dynamics simulations) allowing transport of the hydrophilic phospholipid head group, in a groove outlined by TMs 1, 2, 4 and 6, with the hydrocarbon chains following passively, still in the membrane lipid phase (and presumably outside the channel) — this must disrupt the hell out of the protein as it passes. They call this the credit card model — only the interaction with part of the molecule is important — just as the magnetic stripe is the only important thing about the credit card.