CPN60 Gene Conservancy Via Protein-Sequence Comparison Across
Extremophilic Microorganisms
Richard Barrera
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692.
Microorganisms can survive and flourish in
environments which are detrimental to the
majority of life on the planet. Research
focused on final polypeptide modification in
extremophilic microorganisms, comparing
protein production as in more advanced,
eukaryotic cells. I hypothesize that effective
protein modification-management is an
integral step in early cellular evolution and
that therefore the sample organisms will
demonstrate a high level of cpn60 gene
converservancy; this will provide evidence
for the shared ancestry and interrelatedness
of organisms separated by millions of years
of evolution; my data and recently published
literature also hint at how prion and
neurodegenerative
diseases
may
be
propagated. Specifically, I searched for the
presence of the groEL protein across twelve
sub-classes of extremophiles. The groEL
protein is a 60 kilo-dalton protein, which
makes up the large subunit of the groES/L
complex and is the primary Type I
chaperonin used for final polypeptide
modification and maintenance in thousands
of organisms. I searched for the presence of
the groEL protein, and its exon: cpn60, via
amino acid comparison using the National
Center for Biotechnology Information’s
(NCBI) Basic Local Alignment Search Tool
(BLAST) server. Homo sapiens groEL amino
acid sequence was used as target query in
order to demonstrate similarity between
divergent species. N = 24 extremophiles were
identified as candidates for protein
comparison, selecting one to three members
per sub-class; twenty are sequenced and
obtainable via public record; seventeen
organisms demonstrate ≥ 40% positive
match, fourteen demonstrate ≥ 70% positive
match, and three organisms use a separate
Type
I
chaperonin.
Data
suggests
homologous toroid chambers for facilitation
of
post-translational
polypeptide
modification are extremely prevalent among
the majority of organisms on the planet.
Introduction
It is now known that molecular chaperones
participate in a large variety of cellular
functions. They assist in de novo protein
folding, stabilize proteins under duress and
maintain polypeptide chain components in a
loosely folded state for translocation across
organelle membranes (Kumarevel, et al. 1998).
Research focused on the presence of the groEL
protein, contained in the cpn60 exon; the 60 kD
protein is the large subunit of the groEL/S
complex, in extremophilic microorganisms. The
groEL/S complex is one of the primary Type I
chaperonins used for final polypeptide
modification, and among other responsibilities,
handles
the
folding
of
monomeric
mitochondrial rhodanese (Mendoza, et al.
1991). The target query was the five hundred
and four character sequence of the groEL
protein in Homo sapiens. The target region is
believed to be a universal target of about five
hundred and fifty five bp, and has been found to
be a robust target for species-level
characterization of bacteria, archaea, and
eukaryotes (Hill, et al. 2012). The presence of
the protein sequence among examined
microorganisms illustrates a shared requirement
among divergent species for post-translational
polypeptide modification in order to sustain
basic cellular function. This is significant
because in recent years the scientific
community has discovered life across a
multitude of environments that were heretofore
believed to be uninhabitable: these chaperones,
in conjunction with stress-induced shock
proteins, act as an efficient protein management
system, preventing the aggregation of denatured
proteins within the cell and programmed cell
death. This has led researchers to reconsider the
pervasiveness of life and its ability to adapt,
colonize, and thrive in extraordinarily
demanding environments. Conversely, we are
beginning to understand that prion and
neurodegenerative diseases are often the result
of malfunctioning chaperones within the
cytoplasm or intermitochondrial matrix
respectively, which results in protein
aggregation and cell death (Schon and
Manfredi, 2003).
Materials and Methods
Research was conducted via the following: (1)
Thirty microorganisms across twelve subclasses of extremophiles were identified using
publicly available online databases. (2)
Identified microorganisms were vetted using
the Kyoto Encyclopedia of Genes & Genomes
and GenBank in order to identify whether or
not the complete genome has been sequenced
and published; incomplete sequences are not
available for comparison and were eliminated
from the population. (3) Genome identification
continued until a population of N ≥ 20 was
reached. (4) Amino acid examination was
conducted via protein comparison using the
National
Center
for
Biotechnology
Information’s (NCBI) Basic Local Alignment
Search Tool (BLAST) server. (5) Percentage
conversancy of the amino acid sequence was
calculated between the species using the NCBI
Graphic Representation Tool. (6) A cladogram
was constructed using the NCBI Phylogenetic
Tool in order to visualize the divergence
between species.
Results
Twenty four species of extremophiles across
twelve sub-classes were identified as candidates
for comparison. Of the twenty four species
identified, twenty microorganisms representing
ten groups are sequenced and available on the
National
Center
for
Biotechnology
Information’s database. I could not locate
sequenced organisms belonging to piezophiles
or xerophiles. The targeted amino acid
sequence of the groEL protein was obtained
from Homo sapiens, and contains five hundred
and four characters:
MLRLPTVFRQMRPVSRVLAPHLTRAYAKD
VKFGADARALMLQGVDLLADAVAVTMGP
KGRTVIIEQSWGSPKVTKDGVTVAKSIDLK
DKYKNIGAKLVQDVANNTNEEAGDGTTT
ATVLARSIAKEGFEKISKGANPVEIRRGVM
LAVDAVIAELKKQSKPVTTPEEIAQVATISA
NGDKEIGNIISDAMKKVGRKGVITVKDGK
TLNDELEIIEGMKFDRGYISPYFINTSKGQ
KCEFQDAYVLLSEKKISSIQSIVPALEIANA
HRKPLVIIAEDVDGEALSTLVLNRLKVGLQ
VVAVKAPGFGDNRKNQLKDMAIATGGAV
FGEEGLTLNLEDVQPHDLGKVGEVIVTKD
DAMLLKGKGDKAQIEKRIQEIIEQLDVTTS
EYEKEKLNERLAKLSDGVAVLKVGGTSD
VEVNEKKDRVTDALNATRAAVEEGIVLGG
GCALLRCIPALDSLTPANEDQKIGIEIIKRTL
KIPAMTIAKNAGVEGSLIVEKIMQSSSEVG
YDAMAGDFVNMVEKGIIDPTKVVRTALL
DAAGVASLLTTAEVVVTEIPKEEKDPGMG
AMGGMGGGMGGGMF
Homo sapiens amino acid sequence was
selected for comparison in order to punctuate
evolutionary conservancy between divergent
species. Species were entered into the database,
and seventeen organisms were found to contain
a similar target sequence. The remaining three
organisms, members of the archaea domain,
utilize the DnaJ Type I chaperonin in order to
complete
post-translational
polypeptide
modification. The seventeen species of
extremophiles containing the cpn60 protein
sequence demonstrated a ≥ 40% positive match,
fourteen demonstrated a ≥ 70% positive match,
as shown in Figure 1. Figure 1A gives a graphic
representation of the matching sections of
genome, generated by the NCBI. A visual
representation of genetic divergence is depicted
in Figure 2. The expect value (E-value) was ≤
3.00x10-11.
Discussion
The E-Value represents background noise, or
the percent likelihood that a false positive will
be encountered in the query sequence. The subclasses included in the research were:
acidophiles,
alkaliphiles,
cryptoendoliths,
osmophiles, lithoautotrophs, metallophiles,
oligotrophs,
piezophiles,
psychrophiles,
radiophiles, thermophiles, and xerophiles. This
data is significant because it confirms that life
is predicated on the proper function,
maintenance, and destruction of proteins. Cells
cannot function without a form of intermediary
Identical
Match %
chamber which allows polypeptide chains the
chance to assume , resume, or degrade their
tertiary structures. As such, the evolution of
chaperonins was an integral and promethean
step in the evolution of life on Earth.
Additionally, any chaperonin mutation which
alters its interaction with hydrolysable ATP
binding, or alters the protein-modification
chamber in such a way as to produce a
renegade protein, may result in significant
havoc and ultimately cell death (Walters et al.
2002). For example, research has found that the
malfunctioning of oxidative phosphorylation
pathways in mitochondria leads to the excess
generation of reactive oxygen species. These
species decimate the mitochondria, altering the
structure of whatever they come in contact
with, including chaperonins. These altered
chaperonins can no longer fulfill their duties of
protein maintenance, and as a result, the
mitochondria self-destructs (Mukherjee and
Chakrabarti,
2013).
Positive
Match %
Identical
Match
Expect value
Sphingopyxis alaskensis
56.16
78.36
233
0
Saccharomyces cerevisiae
56.98
75.23
224
0
Wallemia ichthyophaga
56.5
72.74
235
0
Debaryomyces hansenii
56.91
76
231
0
54.7
76.69
238
0
Nitrosomonas sp. AL212
52.37
74.19
250
0
Cupriavidus metallidurans
53.77
74.15
244
0
Acidithiobacillus ferrooxidans
51.04
74.38
257
0
Thiobacillus denitrificans
53.02
73.4
247
1.00E-180
Pelagibacter ubique
Flavobacterium psychrophilum JIP02/86
51.7
73.3
251
1.00E-179
Bacillus subtilis
51.33
73.19
253
1.00E-179
Amphibacillus xylanus
50.57
72.62
257
9.00E-179
48.7
70.26
264
1.00E-163
Pyrolobus fumarii 1A
23.36
44.86
340
1.00E-017
Methanopyrus kandleri AV19
23.23
42.47
323
5.00E-017
Methanococcoides burtonii DSM 6242
23.03
43.07
340
2.00E-015
Sulfolobus solfataricus P2
22.02
41.74
352
3.00E-011
Deinococcus radiodurans
Figure 1. Hit table generated by BLAST data analysis. Percent match is in relation to Homo sapiens template code.
Identical match is number of correct chemical and spatial amino acid matches. Chart is ordered by E Value (most certain
match to least).
Figure 1A. Graphical representation of hit table from Figure 1. Red areas indicate identical matches, grey areas indicate
positive matches. Organisms are listed in the same order as Figure 1.
Figure 2. A phylogenetic tree based on genetic divergence of 504 character amino acid sequence; present are the seventeen
microorganisms sampled which contain the cpn60 gene.