Reply1:Protein localization refers to any process in which a protein complex or an organelle is
transferred by the relevant amino acids and tethered or maintained at its specific
position. Protein localization can also be done through selective degradation of various
proteins. In some proteins, the localization results from the recognition of passively
diffusing protein complexes or soluble proteins (Blobel & Dobberstein, 1975). However,
this form of protein localization is rare because it does not guarantee enough protein
concentrations to maintain some critical processes in the body. Since every protein has
specific loci in the body, delocalization of a protein from its specific loci can result in
detrimental effects on the body. The impacts can be felt at a cellular level, tissue level,
or even in the entire organism. For this assignment, I will propose the localization of the
tumor suppressor gene p53 in the body and the possible associated effects.
Depending on the function of a sequence tag, the secretory pathway plays a
critical role in transferring various proteins to specific cellular compartments. The
transfer process is achieved with the help of a distinctive organelle localization signal
peptide tag or sequence, which is located in the N’ or C’ termini of the various cell
organelles. For example, ER retention signal, KDEL is a signal sequence that initiates
the transfer of various proteins like S-K-L = S-K-L = S-K-L = S-K-L = S-K-L = S from the
Golgi apparatus to the endoplasmic reticulum. Therefore, it is possible to delocalize a
protein to a different cell organelle by removing its native localization signal peptide tag
and replacing it with a peptide tag of other organelle-specific tags (Gallagher et al.,
2004). For instance, the tumor suppressor gene p53 can be a perfect example. It is a
transcription factor, to be precise. As a consequence, it is contained in the nucleus.
When the nuclear localization signal is replaced, it gets localized to the peroxisome, and
the peroxisomal localization signal is included.
The extraction of the tumor suppressor gene p53 from the nucleus, where it
performs most of its function, brings severe consequences to the cell and the entire
organism. For instance, the cell divides massively and abnormally forms an abnormal
tissue (Lacroix et al., 2020). The tissue, on the other hand, develops cancer. If such a
case is not monitored early enough and controlled, the organism ultimately dies due to
metastasis.
Psalms 139 says, “For you knitted me together in my mother’s womb, shaping
my inner pieces. I thank you because you produced me fearfully and wonderfully. Your
works are magnificent; my soul is well conscious of this. When I was being formed in
secret, intricately woven in the depths of the earth, my frame was not concealed from
you” The verses are enough to prove the magnificent and mighty skills God had during
creation (The New King James Version, 1769/2017, Psalms 139). There are many
millions of cell organelles, especially in multicellular organisms, and each one contains
a particular protein that controls its functioning. However, God could place each of them
at a specific place where its functioning is needed. Again, the proteins are established
to coordinate and bring about particular processes in the body. It’s with no doubt that
the works of God are supernatural, and no man can ever match such skills.
References:
•
Blobel, G., & Dobberstein, B. (1975). Transfer of proteins across membranes. I.
Presence of proteolytically processed and unprocessed nascent immunoglobulin
light chains on membrane-bound ribosomes of murine myeloma. The Journal of cell
biology, 67(3), 835-851.
• Gallagher, J. W., Weinberg, R. B., & Shelness, G. S. (2004). apoA-IV tagged with
the ER retention signal KDEL perturbs the intracellular trafficking and secretion of
apoB. Journal of lipid research, 45(10), 1826-1834.
• Lacroix, M., Riscal, R., Arena, G., Linares, L. K., & Le Cam, L. (2020). Metabolic
functions of the tumor suppressor p53: Implications in normal physiology, metabolic
disorders, and cancer. Molecular metabolism, 33, 2-22.
https://doi.org/10.1016/j.molmet.2019.10.002
• The New King James Version, (2017). Biblia.
https://biblia.com/bible/nkjv/psalm/139/13-15. (Original work published 1769).
Read Less
Reply2:
For this discussion, in which a protein was localized to an alternative cellular
compartment, I wanted to suggest and explore the possibility of localizing peroxins to
the outer mitochondrial membrane. The peroxisome has enzymatic actions which
include the synthesis of bile acids and sterols (Terlecky, S.R.et.al.2000-2013). These
organelles tend to be very numerous and can process nitrogenous waste as well as
oxidize very-long-fatty acid chains which are useful cellular features in mammals
(Terlecky, S.R.et.al.2000-2013) The general consensus is that new peroxisomes form
from the splitting or budding of other peroxisomes and that this rate may change with
stress or need (Terlecky, S.R.et.al.2000-2013). Key in the development of peroxisomes
are a protein group called peroxins (Terlecky, S.R.et.al.2000-2013). Approximately 25
peroxins are known with most incorporated into peroxisomal membrane proteins
(PMPs) (Kim, P.K. et.al.2015). Three of the peroxins designated PEX3, PEX16, and
PEX19 are believed to be critical in the formation of the peroxisome while the rest, the
PMP’s, are required for peroxisomal matrix protein import from the cytosol (Kim, P.K.
et.al.2015). Peroxisomal matrix proteins and the peroxisomal membrane proteins are
both nuclear coded and translated on free polyribosomes (Kim, P.K. et.al.2015). These
are a few of the reasons I chose the peroxins as the subject of this experiment because
practically all proteins associated with formation of both the organelle and the
transmembrane machinery are synthesized on free ribosomes in the cytosol (Kim, P.K.
et.al.2015). Membrane-bound ribosomes, such as those on the endoplasmic reticulum,
are in contact with the ER membrane which are efficient for constructing proteins bound
for a secretory pathway. Often these ribosomes make direct contact with a gate in
order to produce proteins, such as transmembrane alpha-helix proteins. For this
experiment, bound ribosomes could add a potential complication of the manipulated
protein getting to the desired target organelle.
Peroxins, and practically all other enzymes marked for the peroxisome, have a
carboxy-terminal targeting signal called peroxisomal targeting signal 1 (PTS1)
(Terlecky, S.R.et.al.2000-2013). Another sequence exists for signaling on a limited
number of enzymes known as PTS2 and attached to the amino terminus when PTS1 is
not present (Terlecky, S.R.et.al.2000-2013).
To get peroxin proteins to localize at the outer membrane of the mitochondria I
propose the use of CRISPR/Cas9 technology (RodriguezRodriguez,D.R.et.al.2019). CRISPR/Cas9 nuclease can target any of the genes that
code for PEX proteins and remove the signal sequence PTS1 or PTS2 dependent on
the specific PEX protein desired (Rodriguez-Rodriguez,D.R.et.al.2019). Both PTS1 and
PTS2 are very short signal sequences with PTS1 only 3 amino acids in length and
PTS2 having a loose consensus of 9 amino acids (Terlecky, S.R.et.al.2000-2013). Once
the peroxisome signal sequence is removed it will need to be replaced with a
mitochondrial targeting sequence. For this thought experiment the targeting sequence
for the outer membrane would be the TOM complex (Alberts, B.,et.al.2002). The
majority of mitochondria proteins are nuclear coded much like the peroxisomal proteins
so I chose this TOM complex because it seemed like the only way peroxisomal proteins
“could” pass through the outer membrane (Alberts, B.,et.al.2002). The TOM complex
makes a good candidate for entry into the outer membrane because it uptakes
precursor mitochondrial proteins directly from the cytosol (Alberts, B.,et.al.2002). Also,
the TOM complex generally requires unfolding of the precursor proteins in order to pass
the membrane (Sato, T.K., et.al.2019). Since folding is not necessarily a prerequisite for
import through PMP’s, there is a possibility that a peroxin could pass through the TOM
complex unfettered (Kim, P.K. et.al.2015). In addition to an added mitochondrial location
signal (MLS), the amino acid sequence correlating to the MLS should be composed in
such a way to create the highest positive charge possible. This allows for the
mitochondrial membrane potential to draw in the altered protein more readily.
If this experiment could recode the signal sequences then a localization of peroxin
proteins could potentially begin building up in the cytosol at the outer mitochondrial
membrane. If the altered proteins were successful in initiating translocation but did not
clear the TOM complex on the intermembrane space, then localization may appear as
dots on the outer membrane as each TOM complex essentially becomes “clogged”. If
the altered proteins succeeded in completely passing the TOM complex then they would
localize and accumulate in the intermembrane space. I would expect they would remain
there because none of the other membrane complexes would recognize those proteins
and thereby be unable to pass them along or back out. I suspect ,in this particular
scenario, that if localization of peroxins to the mitochondria were achieved a two-sided
disruption of cell homeostasis would occur. On one side the recoding of peroxin genes
to target another organelle would deprive the cell of peroxisome factors. This would
prevent the formation of necessary membrane proteins to carry enzymes into the
peroxisome. On the other hand, the disruption of the outer mitochondrial membrane
could prevent uptake of mitochondrial precursor proteins plus a shutdown of the inner
membrane transport pathways. Another potential outcome could be mitochondrial
precursor over-accumulation stress (mPOS) (Coyne, L.P., 2018). There are links
showing that cytosolic proteostatic stress can cause mitochondrial dysfunction leading
to cell death (Coyne, L.P., 2018). Admittedly, this would be an experiment where
targeted organelle dysfunction would be expected and would only have very specific,
real-world applications of therapy, for instance, as a novel, experimental anticancer
treatment.
To comment on God’s creation and complexity of design, when I began researching
for this discussion the complexity of signal sequences and protein trafficking
overwhelmed me. Each time I explored a pathway and began to unravel the multitude
of steps necessary to complete just one synthesis or translocation I was
astounded. Not only the depth of complexity but the interconnectedness of
pathways. It was made clear to me that manipulating any protein pathway had
cascading effects throughout the cell. This intricacy represents just a fractional part of
God’s creative design.
References cited
1. Coyne, L. P., & Chen, X. J. (2018). mPOS is a novel mitochondrial trigger of cell
death – implications for neurodegeneration. FEBS letters, 592(5), 759–775.
https://doi.org/10.1002/1873-3468.12894
2. Hasan, S., Platta, H. W., & Erdmann, R. (2013). Import of proteins into the
peroxisomal matrix. Frontiers in physiology, 4, 261.
https://doi.org/10.3389/fphys.2013.00261
3. Kim, P. K., & Hettema, E. H. (2015). Multiple pathways for protein transport to
peroxisomes. Journal of molecular biology, 427(6 Pt A), 1176–1190.
https://doi.org/10.1016/j.jmb.2015.02.005
4. Terlecky SR, Walton PA. The Biogenesis and Cell Biology of Peroxisomes in Human
Health and Disease. In: Madame Curie Bioscience Database [Internet]. Austin (TX):
Landes Bioscience; 2000-2013. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK6339/
5. Rodríguez-Rodríguez, D. R., Ramírez-Solís, R., Garza-Elizondo, M. A., GarzaRodríguez, M. L., & Barrera-Saldaña, H. A. (2019). Genome editing: A perspective
on the application of CRISPR/Cas9 to study human diseases (Review). International
journal of molecular medicine, 43(4), 1559–1574.
https://doi.org/10.3892/ijmm.2019.4112
6. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New
York: Garland Science; 2002. The Transport of Proteins into Mitochondria and
Chloroplasts.Available from: https://www.ncbi.nlm.nih.gov/books/NBK26828/
7. Sato, T.K., Kawano, S. & Endo, T. Role of the membrane potential in mitochondrial
protein unfolding and import. Sci Rep 9, 7637 (2019).
https://doi.org/10.1038/s41598-019-44152-z
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