To understand anything in the cell you need to understand nearly everything in the cell

Understanding how variants in one protein can either increase or decrease the risk of Parkinson’s disease requires understanding of the following: the lysosome, TMEM175, Protein kinase B, protein moonlighting, ion channel lysoK_GF, dopamine neurons among other things. So get ready for a deep dive into molecular and cellular biology.

It is now 50 years and 6 months since L-DOPA was released in the USA for Parkinson’s disease, and I was tasked as a resident by the chief with running the first L-DOPA clinic at the University of Colorado.  We are still learning about the disease as the following paper Nature vol. 591 pp. 431 – 437 ’21 will show. 

The paper describes an potassium conducting ion channel in the lysosomal membrane called LysoK_GF.  The channel is made from two proteins TMEM175 and protein kinase B (also known as AKT).

TMEM175 is an ion channel conducting potassium.  It is unlike any of the 80 or so known potassium channels.  It  contains two repeats of 6 transmembrane helices (rather than 4) and no pore loop containing the GYG potassium channel signature sequence. Lysosomes lacking it aren’t as acidic as they should be (enzymes inside the lysosome work best at acid pH).  Why loss of a potassium channel show affect lysosomal pH is a mystery (to me at least).

Genome Wide Association Studies (GWAS) have pointed to the genomic region containing TMEM175 as having risk factors for Parkinsonism.  Some variants in TMEM175 are associated with increased risk of the disease and others are associated with decreased risk — something fascinating as knowledge here should certainly tell us something about Parkinsonism.  

The other protein making up LysoK_GF is protein kinase B (also known as AKT). It is found inside the cell, sometimes associated with membranes, sometimes free in the cytoplasm. It is big containing 481 amino acids. Control of its activity is important, and Cell vol. 169 pp. 381 – 405 ’17 lists 21 separate amino acids which can be modified by such things as acetylation, phosphorylation, sumoylation, Nacetyl glucosamine, proline hydroxylation.  Well 2^21 is 2,097,152, so this should keep cell biologists busy for some time. Not only that some 100 different proteins AKT phosphorylates were known as 2017.  

TMEM175 is opened by conformational changes in AKT.  Normally the enzyme is inactive because the pleckstrin homology domain binds to the catalytic domain inhibiting enzyme activity as the substrate can’t get in.

Remarkably you can make a catalytically dead AKT, and it still works as a controller of TMEM175 activity — this is an example of a moonlighting molecule — for more please see — https://luysii.wordpress.com/2021/01/11/moonlighting-molecules/.

Normally the activity and conformation of AKT is controlled by the metabolic state of the cell (with 21 different molecular knob sites on the protein this shouldn’t be hard).  So the fact that AKT conformation controls TMEM175 conductivity which controls lysosome activity gives the metabolic state of the cell a way to control lysosomal function.  

Notice how to understand anything in the cell you must ask ‘what’s it for’, thinking that is inherently teleological. 

Now on to the two risk factors for Parkinsonism in TMEM175.  The methionine –> threonine mutation at amino acid #393 reduces the lysoK_GF current and is associated with an increased risk of parkinsonism, while the glutamine –> proline mutation at amino acid position #65 gives a channel which remains functional under conditions of nutrient starvation. 

The authors cultured dopamine neurons and found out that the full blooded channel LysoK_GF (TMEM175 + AKT) protected neurons against a variety of insults (MPTP — a known dopamine neuron toxin, hydrogen peroxide, nutrient starvation). 

TMEM175 knockout neurons accumulate more alpha-synuclein — the main constituent of the Lewy body of Parkinsonism.

So it’s all one glorious tangle, but it isn’t just molecular biological navel gazing, because it is getting close to one cause (and hopefully a treatment) of Parkinson’s disease.  

Moonlighting molecules

Just when you thought you knew what your protein did, it goes off and does something completely different (and unexpected). This is called moonlighting, and is yet another reason drug discovery is hard. You can never be sure that your target is doing only what you think it’s doing.

Today’s example is PACAP, a neuromodulator/neurotransmitter made by neurons. Who knew that PACAP can and does act as an antibiotic when the brain is infected. [ Proc. Natl. Acad. Sci. vol. 118 e1917623117 ’21 ] does (PNAS no longer pages its journals, as last year’s total was over 33,000 !).   PACAP is a member of the vasoactive intestinal polypeptide, secretin, glucagon family of neuropeptides (mammals have over 100 neuropeptides according to the paper).

PACAP stands for Pituitary Adenylate Cyclase Activating Polypeptide. It comes in two forms containing 27 or 38 amino acids, both cleaved from a 176 amino acid precursor. There are 3 receptors for PACAP, all G Protein Coupled Receptors (GPCRs). A zillion functions have been ascribed to it, setting the circadian clock, protecting granule cells of the cerebellum. Outside the nervous system it is produced by immune cells in response to inflammatory conditions and antigenic stimulation. It is one of the most conserved neuropeptides throughout the course of evolution. Now we probably know why.

Showing how hard protein chemistry really is, PACAP is structurally similar to cathelicidin LL-37 an antimicrobial peptide, despite having less than 5% amino acid sequences in common. PACAP is cationic. Different sides of the protein have different characteristics, with one side being highly positively charged, and the other being hydrophobic (e.g. the protein is amphipathic). This is typical of antimicrobial peptides, and perturbation of microbial membranes by inducing negative Gaussian curvature probably explains its antibacterial activity.

In mouse models of Staph Aureus or Candida infections, PACAP is induced ‘up to’ 50 fold in the brain (or spleen or kidney) where it kills the bugs. Yet another reason drug discovery is so hard. We are mucking about in a system we barely understand.

There are many other examples of moonlighting proteins. Probably the best known is cytochrome c which is is a heme protein localized in the compartment between the inner and outer mitochondrial membranes where it functions to transfer electrons between complex III and complex IV of the respiratory chain. Oxidation and reduction of the iron atom in the heme along with movement along the mitochondrial intermembrane space allows it to schlep electrons between complexes of the respiratory chain.

All well and good. But cytochrome c also can tell a cell to commit suicide (apoptosis) when mitochondria are sufficiently damaged that cytochrome c can escape the intermembrane space. Who’d a thunk it?

How many more players are there in the cell (whose function we think we know) that are sneaking around — doing more things in heaven and Earth, Horatio, than are dreamt of in your philosophy?