Hyperlipid by Petro Dobromylskyj

18 April 2019

You need to get calories from somewhere, should it be from carbohydrate or fat?
  • Life (31) Chinese whispers
    Back in 2008 Noha Mesbha published her excellent PhD thesis

    ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND PHYSIOLOGICAL ADAPTATIONS TO MULTIPLE EXTREME CONDITIONS

    which introduced the world, via this paper

    The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

    to Natranaerobius thermophilus and its antiporter nt-Nha. Which gives every impression of being a stand alone Na/H+ antiporter very closely related to the invariably operon controlled MrpA protein (named shaA) of Clostridium tetani. As she says

    "Gene nt-Nha had 35% identity to the shaA (mrpA) gene of Clostridium tetani. The Mrp proteins belong to the monovalent cation/proton antiporter-3 protein family....Sequence analysis of the regions surrounding gene nt-Nha, however, did not show that it was part of an operon. This indicates that gene nt-Nha does not encode a subunit of an Mrp system, but rather a mono-subunit antiporter".

    All well and good.


    Then in 2010 Morino et al published

    Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

    Although the whole paper was about B subtilis (and how none of its Mrp subunits worked in any incomplete combination to antiport anything) they did make this throw-away comment:

    "A MrpA/MrpD homologue encoded by a “stand alone” gene from polyextremophilic Natranaerobius thermophilus was recently reported to exhibit Na+/H+ and K+/H+antiport activity in anaerobically grown E. coli KNabc (24)"

    where (24) is Mesbha's PhD paper. Notice that at this stage Mesbha's

    nt-Nha ~ shaA, very closely related at 35% conserved gene sequence,

    has been changed by Morino in to

    nt-Nha ~ An "MrpA/MrpD homologue".

    This is a just about acceptable per se because MrpA and MrpD are homologous to each other and nt-Nha is closely related to MrpA (shaA) of C tetani. But Mesbha herself never mentions MrpD in her 2009 paper or in her PhD thesis. And "MrpA/MrpD" is open to mis-interpretation. So we have "definition-creep" here, where nt-Nha could be accidentally seen as some sort of composite of MrpA in combination with MrpD. Ouch.

    So next we have the 2017 offering by Jasso-Chávez et al

    Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans

    where we have this bizarre statement

    "On the other hand, a fused MrpA-MrpD homolog in the alkaliphilic Natranaerobius thermophilus displayed Na+/H+ antiport activity when produced in E. coli strain KNabc (5, 28)"

    Ref (5) is Morino's paper on B subtilis Mrp, in which one rather misleading citation suggests that nt-Nha is an "MrpA/MrpD homologue". This has developed to the extent that nt-Nha has now "become" a fusion of two genes to give a rather mythical monster.

    Ref (28) is just Mesbha's PhD paper in which nothing of the sort was suggested.

    So the Jasso-Chávez paper is utterly flawed due to misinterpreting a poorly phrased statement and adding an erroneous modification so as to grossly mis-represent an initial very solid finding by Mesbha. The Jasso-Chávez discussion of nt-Nha can be distilled as:

    "Send three and fourpence, we're going to a dance".

    The chance of their understanding how nt-Nha or their very own archaeal MrpA subunit work as a stand-alone antiporter appears to be approximately zero.

    Very sad.

    Peter

    BTW The folks who worked from the actual gene to model the nt-Nha protein structure suggest that

    "The final model presents 13 transmembrane α-helices organized in a similar arrangement as the NuoL subunit".

    You know the picture but here it is again 'cos I think it's lovely






  • To the gym?
    This press release in advance of a poster presentation is doing the rounds on social media at the moment:

    Ability to lift weights quickly can mean a longer life

    A factual association is probably true between being above the median in ability to do work on a specifically selected gym machine and longevity.

    What does this mean? It's observational. Hypothesis generation only.

    Perhaps non-athletes with higher power output might live longer because they have excellent mitochondria and our mitochondria are essentially what determine our longevity. Having good mitochondria might well mean that you exercise spontaneously without describing yourself as an athlete. As in I might carry a couple of sacks of chicken food myself rather than wait for Paul the yard-man and his barrow. But this is an effect, not a cause.

    Non-athletes with low power output may be the converse. They have mitochondria in which the cardiolipins anchoring their cytochrome-C to the mitochondrial inner membrane are as flimsy as a PUFA in a deep fat fryer, which are willing to trigger apoptosis at the drop of a superoxide molecule. Poor mitochondria, less muscle fibres giving sarcopaenia, shorter longevity. Probably already giving diabetes in-situ. An AHA poster-child.

    Taking someone from the second category and making them exercise might convert them to being healthy with a long life span. Maybe.

    Or, there again, it might make the gym sessions so unbearable that they quit.

    Or, if you don't let them quit, it might simply make no difference.

    Or, if you don't let them quit, it might kill them sooner!

    As Eeyore said "think of all the possibilities, Piglet, before you settle down to enjoy yourselves".

    The study (as per press release) tests nothing. Researchers are giving very specific advice based on an untested observation which might be going to make them look very, very stupid when the results of an intervention to test their hypothesis actually refutes it. But that will take decades and hopefully the researches will be retired by then. More likely it will be ignored or termed a paradox. All the possibilities Piglet.......

    EDIT or the intervention might show benefit. That's not impossible! END EDIT

    Peter

    Declaration: I have nothing against exercise. Nowadays I mostly boulder because it is three dimensional problem solving using muscle groups to failure on a regular basis. Plus I also try to keep my cardiolipins as saturated as practical!
  • Life (30) Guesses about Na+ channels
    Complex I exists in various states, the two main ones being activated and deactivated. The deactivated form is convincingly a Na+/H+ antiporter. There is a pretty good case made in this paper, which has a number of flaws but is generally probably correct:

    The deactive form of respiratory complex I from mammalian mitochondria is a Na+/H+ antiporter

    As always, the suspect antiporter is thought to be NuoL, the distal "antiporter-like" subunit. You can see the logic for this which is supported by the stand-alone antiporting homologues of NuoL seen in nt-Nha and at least one archaeal MrpA subunit. Personally I'm not sure this is the case. The phenomenal difficulty in trying to interpret exactly what is happening in a structure as intricate and as minute as complex I allows many views of the available data.

    It also appears to be the case that in bacteria which use menaquinone as their electron acceptor (rather than CoQ) some degree of Na+/H+ antiporting occurs under normal active NADH oxidation/proton pumping. That's in here

    Respiratory complex I: A dual relation with H(+) and Na(+)?

    and here's the energetics doodle from the same Fig 2 as I pinched the NADH:ubiquinone doodle from in the last post:













    Because transferring two electrons from NADH to menaquinone only provides 480mV/2e- the complex uses Na+ moving down its concentration gradient to "top up" energy availability and so get the extra energy needed to pump the full four protons.

    Aside: The paper has lots of good ideas but they are very wedded to the concept that NuoL, M and N are still antiporters and that loss of "control" by the redox cytoplasmic arm allows this antiporting to re establish. It's a very reasonable idea but I think it can be improved upon, especially now we have more detailed information about the Na+ pumping of the P furiosus MBH, where this is not what has happened. End aside.

    Why on earth should it matter whether complex I pumps the full complement of four protons? If there is only enough Gibbs free energy for two or three protons, why not just pump two or three protons?

    What occurred to me is that for complex I to pump the four protons it might be necessary to have a full "priming" of the membrane arm with enough Gibbs free energy for a full "push to the left", as in this doodle, discussed in a previous post:























    What if you only have 480mV/2e- available, giving a half hearted "nudge" to the left when you need the full shove from 840mV/2e-? Is it possible that, under these circumstances, nothing at all happens? There is no flip of the glutamate/lysine pairs from together to apart, which triggers all of the opening/closing of water channels that allows proton translocation? Zero proton translocation?

    If you wanted to restore the full "kick to the left" it might be a reasonable energetic top-up to supply the extra energy from Na+ ingress up near the Q binding site to allow the menaquinone plus Na+ ingress to generate the full Gibbs free energy for activation of the membrane arm. That needs a trans-membrane channel, so we are looking at the really complicated region around NuoH and NuoA/J/K. Not easy.

    The best characterised Na+ channel in the whole related set of the complex I, MRP and MBH systems is the one in the MBH of P furiosus, described by Yu et al in

    Structure of an Ancient Respiratory System

    which gives the Na+ channel looking like























    with the red blobs being the modelled Na+ binding sites. This is made up of proteins from four separate genes and is thought to be homologous to the Na+ channel of the MRP antiporter, also a multigene structure. They show them as identical in their discussion doodles, like this for the MRP multigene Na+ channel, simplified to the subunit shown as MrpG:














    The equally multigene Na+ channel of the MBH of P furiosus (in this doodle the multiple Mbh genes are simplified to MbhC) is shown in exactly the same location with the same critical broken helix, in the same shade of green:

















    The final doodle in this figure is complex I. Here is their image:













    which keeps things nice and simple between NuoH and the Nuo A/J/K region, where Nqo10 is shown but nothing else. So I wanted to know if there could be a Na+ channel which could be used to either top up the energy of the NADH:menaquinone couple or to allow the antiporting function to occur in deactivated standard CoQ based complex I. Of course no one is looking for Na+ channels in complex I in quite the same way as Yu et al were looking for one in MBH, where they knew it was crucial. Anyway, I went looking. In here

    Structure and function of mitochondrial complex I

    is where I found find this image (which is unfortunately left to right transposed in its view) that includes a rather nice broken helix shown in pink which I have circled in red:

















    If we take this broken helix, colour it green and transplant it in to Yu et al's complex I we get this:













    Might it be the Na+ channel I need?  No one knows (yet). It is known that there is a conformational change in the region of the CoQ binding pocket when complex I changes from the active to the deactivated state, close to the region of the broken helix head. I would suggest this conformational change might allow the Na+ antiporting function to occur when pumping in complex I is deactivated. Protons would be allowed to enter the cell/mitochondrion in exchange for Na+ expulsion (theoretically reversible but that seems unlikely physiologically unless you are a bacterium using menaquinone where some Na+ ingress is worth it to trigger the membrane arm). Logic says that this trade off would be preserved if antiporting provided a net benefit to the organism under which ever conditions might have favoured deactivation of complex I.

    Peter

    Addendum. Here are the three complexes lined up. Because PowerPoint lets you do it. No other reason.

















  • Life (29) Applied billiards to MRP and MBH
    Here is a nice doodle of complex I taken from this interesting but off topic paper














    It shows that taking two electrons from NADH and dropping them to CoQ provides a Gibbs free energy of 840mV for the pair. That's all well and good and is why cells love oxygen based electron transport chains. But I'd like to look at this in reverse. We know that complex I works perfectly well in reverse. So we can say that dropping four protons from extracellular to intracellular down a membrane voltage of 150mV across a proton tight membrane will allow the performance of 840mV of work.

    For complex I running in reverse this 840mV of work is always used for NADH generation from NAD+ (or superoxide generation) because that's the unit bolted on to the membrane arm. Other metabolic units can be used in place of NADH dehydrogenase and many of these can be found in various bacteria using various substrates with various numbers of antiporter-like subunits in the membrane arm. All will work in reverse and provide/utilise differing Gibbs free energies.

    So I would like to view the membrane arm of all of these complexes as a machine in its own right which, when running in reverse, can harvest the energy available from a proton gradient for whatever function the cell might like. Obviously this is not the current arrangement, but is possible.

    So here is the intact membrane arm of the complex I of T thermophilus, once again taken from Luca et al












    running in reverse to give 840mV/2e- of usable energy. As in the last post we can imagine each proton passing through each channel "kicks" to the right ('cos we're running in reverse here) until the cumulation of four "kicks" is enough to cause the conformation change in Nqo 8 which drives two electrons back up the FeS chain to an NAD+. Think of the membrane arm as a machine to accumulate multiple "kicks" to give a big Gibbs free energy.

    Now let's remove some of the sub units and see what we get. How about this:












    Two proton translocations will provide much less Gibbs free energy than four proton translocations. I've faintly allowed a third proton channel but there is no suggestion this is a functional feature of complex I at all. It's Nqo 10, never mentioned in the paper. Note that all of the subunits are kicking in the same direction.

    The above is obviously the basis of the MRP antiporter, which looks like this:



















    where the energy of two protons is cumulated to allow a single Na+ ion to be extruded. How much of a Gibbs free energy will be developed by the two protons will obviously be influenced by the membrane voltage through which the protons fall. It will also be influenced by the permeability to protons of the membrane in which MRP is embedded. If the membrane was 100% permeable to protons there is no point trying to harvest a gradient which isn't there. The more permeable the membrane the less energy can be harvested per proton. But we do know that in E coli under CCCP with a membrane voltage of as little as 15mV that MRP can cumulate enough Gibbs free energy to antiport Na+ when monogenetic antiporters fail completely.

    In the ancestral hydrothermal vent scenario a membrane voltage from the pH gradient between vent and oceanic fluids might be, ideally, around 150-200mV. I still consider MRP is most likely an adaptation to allow colonisation of vent environments where apposition of fluids is not perfect or some mixing has already occurred and the available membrane voltage might be very low. Given MRP LUCA might survive on the edges of vents in addition to thriving in the luxurious conditions of perfect apposition near the centre.

    A step on from MRP is, hypothetically, to add a power unit which will allow Na+ pumping when membrane voltage is too low even for MRP. You can think of this as an adaptation to the tail end of the vent system. Some membrane voltage is still present as are complex molecules (from dying prokaryotes in the better vent environments) out of which ferredoxin can be synthesised. This is not quite the membrane bound hydrogenase of P furiosus as we will see. It's an augmented "kick-to-the-right" through MRP to assist the generation of enough Gibbs free energy to allow Na+ extrusion:























    Notice that all of the "kick" arrows run in the same direction as for all of the illustrations in this post, looking to generate a significant Gibbs free energy at the right hand end.

    The final stage of generating the MBH of P furiosus (and freedom from the vent gradient completely) is like this: We have to reverse the direction of the proton travel in MBH subunit H. The "kick" from proton ingress is to the right. We want to impose a kick towards the left to reverse the proton flow to give egress, using ferredoxin power. This requires a barrier between MbhM and MbhH to stop that kick to the right, completely different to complex I and any doodle so far. As Yu at al comment in the legend to Figure 3

    "A hydrophilic axis across MbhM membrane interior is also identified but it is separated from that in MbhH due to a gap between the two subunits"

    and in the legend to Figure S4:

    "Note the large gap between subunits M and H in MBH (A). There are four elongated densities located to the lower region of the gap (A) inset; marked by blue dashed lines), which stack against several hydrophobic regions of subunits M and H. These densities are likely from two phospholipid molecules that may stabilize the structure and prevent ion leakage across the membrane bilayer. The dashed curves in (C) and (D) highlight the fact that the chain of hydrophilic residues found in complex I is continuous (D), but is discontinuous in MBH (C)".

    This is the genuine article:















    and my version, more crudely:























    As well as stopping MbhM "kicking" the central water channel protons of MbhH to the right, the conformation change from the NiFe hydrogenase has to be imposed on MbhH to force a proton outwards.

    So the white arrow of proton "kick" in the central water channel of MbhH is reversed by the green cranks using the power from the hydrogenase (green arrow) to enforce this. The bright red proton channel is present as specified by Yu et al and is both essential and functional. Quite what the remnant of this is doing complex I I don't know and quite what the proton channel within MbhM is preserved for I also can't imagine (yet). But the general principles of proton movement as set down here are much more satisfying than my initial thoughts on MRP and MBH.

    Peter
  • Life (28) Proton Billiards
    Here we are looking at complex I from from Thermus thermophilus as featured in this paper:

    Symmetry-related proton transfer pathways in respiratory complex I

    Because it's bacterial it is all labelled up in Nqo terminology. In mammalian complex I we use Nuo terms, where NuoH is Nqo8, NuoL is Nqo 12, NuoM is Nqo 13 and NqoN is Nqo 14. We can ignore all of the other subunits.

    Here are the water channels used to allow protons to move through complex I, red beads being water molecules:









    The CoQ binding site (and the NADH dehydrogenase unit) are at the right hand end. What is most important is that the water channels are not all open (hydrated) at the same time. In the resting state the N-side channels are open. A conformational change in Nqo8 is induced by CoQ reduction which opens its water channel and allows a proton to enter from the cytoplasm. This triggers a chain of conformational changes horizontally along the central water channel, moving a proton from right to left within each antiporter-like subunit and which also closes the N-side water channels and opens the P-side water channels, to allow protons to move outwards in to the periplasm. This is their doodle from the discussion:























    If anyone goes through the diagram in the sort of detail I did they can see that Nqo13 doesn't make sense because glutamic acid E377 is not a lysine (K abbreviation), which it is in the other two subunits. That messes up all of the charge movements and the inter-subunit electrostatic binding. From Fig 1 section B elsewhere in the paper you can see there is an arginine (R163) just "north" of E377 which might be doing this job by binding to the aspartate (D166) of Nqo12 but I can't see that this is addressed anywhere in the paper. So it's just my guess. Still. The basic concept is pretty convincing.


    TLDR: The reduction of CoQ to CoQH2 clunks protons horizontally within the central hydrated channel of each antiporter-like subcomplex from their input zone to their output zone.


    We have to bear several things in mind. First is that the system is completely reversible today. As in reverse electron transport using a high membrane potential and reduced CoQ couple to reduce NAD+ to NADH, and generate ROS when NAD+ is all used up... Protons will move inwards from periplasm to cytoplasm as this happens.

    Also this is complex I, it is a relatively late addition to modern bacterial metabolism dependent on proton tight membranes and the availability of molecular oxygen.

    Third is that our best remnant of LUCA is the Na+ pumping membrane bound hydrogenase of P furiosus and this drives from left to right through an Nqo14/NuoN related subunit (and will almost certainly be equally reversible) to its Na+ channel.

    I have some Powerpoint doodles to take this a little further.

    Peter