Allotopic expression and other fun stuff was RE: specific amino acid restriction does the same thing as calorie restriction?

From: Rafal Smigrodzki (rafal@smigrodzki.org)
Date: Tue Apr 22 2003 - 13:17:13 MDT

  • Next message: Rafal Smigrodzki: "RE: specific amino acid restriction does the same thing as calorie restriction?"

    Robert wrote:

    > On Mon, 21 Apr 2003, Rafal Smigrodzki wrote:
    >
    >> ### Do you think that protein aggregation is causative in sporadic
    >> AD or PD, or that it is involved in the pathomechanism at some later
    >> stage, perhaps amplifying the damage caused by other factors?
    >
    > Hard to say -- I'm not that familiar with either the AD or PD
    > literature (once I knew a bit more but that has long since fallen out
    > of my brain).

    ### The debate on whether protein aggregation is causal in AD and PD is
    raging as we speak. I am on the side of doubters, and I think misaggregation
    merely amplifies other problems, mainly the accumulated mtDNA mutations.

    ---------------------------------

    >
    >> Recently it turned out that APP has a direct toxic effect on
    >> mitochondria, and mutant alpha-synuclein has also been implicated in
    >> diminished mito function.
    >
    > I could see some strange protein aggregation function clogging up the
    > mitochondrial transport proteins -- APP is only a few dozen amino
    > acids I think. The mitochondrial transport proteins have to have
    > pretty darn large pore sizes to get all of the mito proteins inside.

    ### There are four different mitochondrial protein transport systems. Quite
    complex, yes.

    ----------------------------------

    >
    > If that is the case -- or some other process degrades mitochondrial
    > function then it seems likely that one will get either a shortage
    > of ATP (that's bad for protein synthesis *and* breakdown) or perhaps
    > the production of more free radicals (though improper protein
    > function)

    ### Indeed, there is diminished complex IV activity in AD (discovered by my
    boss, Davis Parker), and increased production of free radicals, although I
    do not know if diminished levels of ATP have been observed as well.

    ---------------------------------
    >
    >> Also, are protein aggregates in AD and PD really lysosomal, or
    >> cytoplasmic (or even extracellular)?
    >
    > A think APP starts out cytoplasmic but is supposed to get exported to
    > to the cell membrane or even outside the cell but a lot can happen to
    > mutant proteins on the way to the forum. E.g. if the mutation is in
    > part of the signal sequence controlling transport and ultimate
    > destination things could get really screwed up. [I think I've heard
    > George Martin state that there is a polymorphism in the signal
    > sequence (not perhaps a "mutation" per se) of the Werner's Syndrome
    > protein -- that might have a very interesting effects by impacting
    > the rate of transport of the newly synthesized protein into the
    > nucleus and therefore the quantity available impacting the rates of
    > DNA copying, transcription, etc. (it isn't fully clear I think
    > exactly when and where the WS protein is functioning) and further
    > downstream the overall "rate" of aging.]

    ### I meant some scepticism as to the usefulness of improving the digestion
    of protein aggregates by lysosomes. AFAIK, AD and PD are not really
    lysosomal disorders, although in PD there is indeed some impairment of the
    proteasome.
    ----------------------

    > What is an Atheroma?

    ### Accumulation of fatty, cholesterol-laden material.

    -------------------------------

    >
    >> Is there any indication that lipofuscin is indeed causally involved
    >> in cell death?
    >
    > I'm not so sure it is "cell death" -- sufficient #'s of double strand
    > breaks will activate the apoptosis progam (and human cells are a lot
    > less tolerant of double strand breaks than mouse cells -- perhaps a
    > major reason they get cancer sooner even though they have many fewer
    > cells).

    ### Apoptosis is a final common fate for a number of processes. The
    apoptosis we observe in PD cybrids is not related to double strand breaks.
    Same applies to apoptosis in intact tissue, although the process as only
    recently been observed.

    -----------------------------------

    >
    > Cell death *is* part of the process -- those cells that decide to
    > undergo apoptosis had better be replaced by stem cells -- otherwise
    > you are just going to fade away. I suspect there is a delicate
    > balance between underactive stem cells -- so you don't heal as fast,
    > gradually lose functional capacity, etc. and overactive stem cells --
    > potentially posed to turn into various forms of cancer.
    >
    > The data, for at least many cell types, I believe suggests that
    > lipofuscin accumulates with age -- thus the recycling ability of
    > lysosomes containing lipofuscin (or perhaps cytoplasmic lipofuscin)
    > presumably declines and the cells may compensate by making more
    > lysosomes -- thus the cells fill up with lysosomes containing
    > lipofuscin (or perhaps merely lipofuscin itself). This is supported
    > by the fact that the cells effectively lose water content with age
    > (more lipofuscin, more lysosomes = less water). So if the cells are
    > filling up with lipofuscin/lysosomes there is less and less space for
    > the mitochondria that you need to generate the energy the cell
    > requires -- thus "Energy Catastrophe". The increasing density of the
    > cell probably also slows down transport processes to the cell surface
    > and/or protein export. That certainly has to have some negative
    > consequences.

    ### PD and AD cybrids have impaired mito function despite never having any
    of the lipofuscin transferred during their production, so it appears that
    the basic defect is in the mitos themselves, although one cannot exclude the
    possibility that lipofuscin makes the whole situation worse.

    ------------------------
    >
    > I had the mtDNA-transfer to the nucleus idea probably 7-8 years ago. I
    > don't remember whether I came up with it. It may have come from a
    > theoretical paper that I read. Aubrey de Grey I think figured it out
    > independently and has worked on it a bit more and I think he thinks
    > its a bit tricky because the human mtDNA proteins that are currently
    > still in the mtDNA are a bit hydrophobic and so transport and getting
    > them across the membranes may be difficult.
    >
    > I think he has some possible work-arounds -- do a PubMed search for
    > his papers over the last 3-4 years or send him an email.

    ### Yes, I checked his bibliography.

    The idea of allotopic mtDNA expression has apparently been around since the
    1980's.

    There is one cool way of solving the problem:

    Entelis N. Kolesnikova O. Kazakova H. Brandina I. Kamenski P. Martin RP.
    Tarassov I. Import of nuclear encoded RNAs into yeast and human
    mitochondria: experimental approaches and possible biomedical applications.
    Genetic Engineering (New York). 24:191-213, 2002.

    Mitochondria import from the cytoplasm the vast majority of proteins and
    some RNAs. Although there exists extended knowledge concerning the
    mechanisms of protein import, the import of RNA is poorly understood. It was
    almost exclusively studied on the model of tRNA import, in several
    protozoans, plants and yeast. Mammalian mitochondria, which do not import
    tRNAs naturally, are hypothesized to import other small RNA molecules from
    the cytoplasm. We studied tRNA import in the yeast system, both in vitro and
    in vivo, and applied similar approaches to study 5S rRNA import into human
    mitochondria. Despite the obvious divergence of RNA import systems suggested
    for different species, we find that in yeast and human cells this pathway
    involves similar mechanisms exploiting cytosolic proteins to target the RNA
    to the organelle and requiring the integrity of pre-protein import
    apparatus. The import pathway might be of interest from a biomedical point
    of view, to target into mitochondria RNAs that could suppress pathological
    mutations in mitochondrial DNA. Yeast represents a good model to elaborate
    such a gene therapy approach. We have described here the various approaches
    and protocols to study RNA import into mitochondria of yeast and human cells
    in vitro and in vivo.

    -------

    Also, at least for some proteins the mito import is not a problem:

    Guy J. Qi X. Pallotti F. Schon EA. Manfredi G. Carelli V. Martinuzzi A.
    Hauswirth WW. Lewin AS.
    Rescue of a mitochondrial deficiency causing Leber Hereditary Optic
    Neuropathy.[comment].
    Annals of Neurology. 52(5):534-42, 2002 Nov.

    Abstract
    A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I
    was the first point mutation in the mitochondrial genome linked to a human
    disease. It causes Leber Hereditary Optic Neuropathy, a disorder with
    oxidative phosphorylation deficiency. To overcome this defect, we made a
    synthetic ND4 subunit compatible with the "universal" genetic code and
    imported it into mitochondria by adding a mitochondrial targeting sequence.
    For detection we added a FLAG tag. This gene was inserted in an
    adeno-associated viral vector. The ND4FLAG protein was imported into the
    mitochondria of cybrids harboring the G11778A mutation, where it increased
    their survival rate threefold, under restrictive conditions that forced the
    cells to rely predominantly on oxidative phosphorylation to produce ATP.
    Since assays of complex I activity were normal in G11778A cybrids we focused
    on changes in ATP synthesis using complex I substrates. The G11778A cybrids
    showed a 60% reduction in the rate of ATP synthesis. Relative to
    mock-transfected G11778A cybrids, complemented G11778A cybrids showed a
    threefold increase in ATP synthesis, to a level indistinguishable from that
    in cybrids containing normal mitochondrial DNA. Restoration of respiration
    by allotopic expression opens the door for gene therapy of Leber Hereditary
    Optic Neuropathy
    -----

    Actually, only two mt proteins have *not* been found in the nucleus in one
    organism or the other. Possibly the reason for stability of the mtDNA in
    evolution is not the protein import problem, but the difference in the
    nuclear and mito genetic code. Even if your cyt b gene is transferred to the
    nucleus (as happened many times, even in humans, as shown by nuclear mito
    pseudogenes), it will be transcribed into a non-functional protein because
    the interpreting machinery (the tRNA code) is different.

    ----------------------------------
    >
    > The short summary is that there are a number of ways to tackle
    > this problem. The pain in the rear end problem that we really
    > need to solve is what the heck in the mitochondrial transport
    > chain is generating the free radicals in the first place.
    > Every time I hear a talk on this topic it gives me a headache
    > due to the complexity.
    >
    ### Me too.

    Rafal



    This archive was generated by hypermail 2.1.5 : Tue Apr 22 2003 - 10:27:12 MDT