Proteomics: Applications and Advances 18.6 Natural Products 487
Part C | 18.6
could assist in identifying novel bioactive proteins, proteomic techniques have not been utilized to any great
extent in marine bioprospecting [18.4]. Nevertheless,
there are a few studies which have employed a proteomic approach to identify or investigate bioactive
products from marine organisms.
Evans et al. [18.91] employed iTRAQ as a means
of characterizing and quantifying expression of the secretome of the marine bacterium Pseudoalteromonas
tunicate. The bacterium, which is associated with marine eukaryotes, produces a broad spectrum of extracellular compounds that are active against invertebrate
larvae, algal spores, diatoms, fungi, heterotrophic flagellates, and bacteria, and are thought to provide the
bacterium with a competitive advantage in mixed communities [18.91]. The study compared the wild-type
secretome to that of a transposon mutant that was no
longer pigmented and did not produce the bioactive
compounds as a consequence of a disrupted type-II secretion pathway. Evans et al. [18.91] found a range of
cytosolic proteins and an ABC-transporter (ABC: ATPbinding cassette) that were significantly differentially
expressed between the two strains. They were unable to
determine the functions of 23% of the proteins comprising the Pseudoalteromonas tunicate secretome, and
suggested that the type-II secretion pathway may play
a role in iron uptake. The authors make the point that
the data obtained from this study will lead to a better understanding of the regulation of pigmentation and
production of the bioactive compounds in Pseudoalteromonas tunicate, as well as elucidate the strategies
the bacterium uses to eliminate competitors and survive as a surface-associated bacterium in the marine
environment [18.91].
It is essential that macromolecular targets of small
bioactive molecules are identified in order to understand their mechanism of action and develop new
pharmaceuticals. Margarucci et al. [18.92] used chemical proteomics [18.93] to investigate the target of
petrosaspongiolide M (PM), a -hydroxy butenolide
marine terpenoid isolated from the sponge Petrosaspongia nigra that has anti-inflammatory properties. The
protocol involved chemically immobilizing PM by covalently linking the molecule to solid beads which
are subsequently incubated with cell lysates. Proteins
anchored to the beads are released, resolved by SDS-PAGE, and finally identified by LC MS/MS analysis [18.92]. The protein mixture eluted from the beads
included a number of proteins known to comprise the
ubiquitin–proteasome system responsible for degrading proteins involved in the cell cycle, apoptosis, signal
transduction, and the NF-ġ (nuclear factor) pathway.
Since abnormal expression of the ubiquitin–proteasome
system is responsible for many human diseases such
as cancer, inflammation, cardiovascular diseases, and
neurodegenerative disorders, Margarucci et al. [18.92]
concluded that the ability to block proteasome activity using PM suggests that the molecule has great
potential as a new lead therapeutic agent. Chemical proteomics was also used in two independent studies that
showed that the eukaryotic translation initiation factor
eIF4A is the target of pateamine A from the marine
sponge Mycale sp., which is a potent cytotoxin against
rapidly growing cells (such as P388 leukaemia cells;
IC50 0:15 ng=ml [18.94]) [18.95, 96].
The genus Conus is comprised of 700 species of
cone snails, which are carnivorous marine gastropods
that use venom for defence, competition, and predation [18.97–100]. Their venoms, termed conotoxins or
conopeptides, have been extensively studied due to the
number and diversity of the neuropharmacologically
active peptides they contain (50 to >1000 per species),
which specifically target a broad range of therapeutically relevant receptors and ion channels [18.97, 99,
100]. Indeed, the drug Ziconotide (Prialt), derived from
the venom of Conus magus, has been approved for
treating severe chronic pain [18.101, 102]. A number
of proteomic studies have been conducted to characterize the venom gland peptidome and proteome of
cone snails in order to increase our understanding of
conotoxin biosynthesis, maturation, secretion, and interspecies variation. A total of 161 and 157 proteins and
protein isoforms were identified in the venom glands
of Conus novaehollandiae and Conus victoriae, respectively, with a large fraction of the proteins found to
function in protein/peptide translation, folding, and protection events [18.99].
In another study, Tayo et al. [18.97] used an LTQOrbitrap tandem mass spectrometer to investigate the
venom proteome in the proximal, central, and distal
portions of the tubular venom duct of Conus textile
in order to elucidate the overall envenomation strategy
employed by Conus species. 31 conotoxin sequences
and 25 post-translational modification variants were
identified following searches using SEQUEST (tandem mass spectrometry data analysis program used for
protein identification) and ProLuCID (name of a tandem mass spectrabased protein identification program)
for unmodified and post-translationally modified peptides, respectively. The study found both qualitative
and quantitative differences in the conotoxin components sampled from the different sections of the
Part C | 18.6
could assist in identifying novel bioactive proteins, proteomic techniques have not been utilized to any great
extent in marine bioprospecting [18.4]. Nevertheless,
there are a few studies which have employed a proteomic approach to identify or investigate bioactive
products from marine organisms.
Evans et al. [18.91] employed iTRAQ as a means
of characterizing and quantifying expression of the secretome of the marine bacterium Pseudoalteromonas
tunicate. The bacterium, which is associated with marine eukaryotes, produces a broad spectrum of extracellular compounds that are active against invertebrate
larvae, algal spores, diatoms, fungi, heterotrophic flagellates, and bacteria, and are thought to provide the
bacterium with a competitive advantage in mixed communities [18.91]. The study compared the wild-type
secretome to that of a transposon mutant that was no
longer pigmented and did not produce the bioactive
compounds as a consequence of a disrupted type-II secretion pathway. Evans et al. [18.91] found a range of
cytosolic proteins and an ABC-transporter (ABC: ATPbinding cassette) that were significantly differentially
expressed between the two strains. They were unable to
determine the functions of 23% of the proteins comprising the Pseudoalteromonas tunicate secretome, and
suggested that the type-II secretion pathway may play
a role in iron uptake. The authors make the point that
the data obtained from this study will lead to a better understanding of the regulation of pigmentation and
production of the bioactive compounds in Pseudoalteromonas tunicate, as well as elucidate the strategies
the bacterium uses to eliminate competitors and survive as a surface-associated bacterium in the marine
environment [18.91].
It is essential that macromolecular targets of small
bioactive molecules are identified in order to understand their mechanism of action and develop new
pharmaceuticals. Margarucci et al. [18.92] used chemical proteomics [18.93] to investigate the target of
petrosaspongiolide M (PM), a -hydroxy butenolide
marine terpenoid isolated from the sponge Petrosaspongia nigra that has anti-inflammatory properties. The
protocol involved chemically immobilizing PM by covalently linking the molecule to solid beads which
are subsequently incubated with cell lysates. Proteins
anchored to the beads are released, resolved by SDS-PAGE, and finally identified by LC MS/MS analysis [18.92]. The protein mixture eluted from the beads
included a number of proteins known to comprise the
ubiquitin–proteasome system responsible for degrading proteins involved in the cell cycle, apoptosis, signal
transduction, and the NF-ġ (nuclear factor) pathway.
Since abnormal expression of the ubiquitin–proteasome
system is responsible for many human diseases such
as cancer, inflammation, cardiovascular diseases, and
neurodegenerative disorders, Margarucci et al. [18.92]
concluded that the ability to block proteasome activity using PM suggests that the molecule has great
potential as a new lead therapeutic agent. Chemical proteomics was also used in two independent studies that
showed that the eukaryotic translation initiation factor
eIF4A is the target of pateamine A from the marine
sponge Mycale sp., which is a potent cytotoxin against
rapidly growing cells (such as P388 leukaemia cells;
IC50 0:15 ng=ml [18.94]) [18.95, 96].
The genus Conus is comprised of 700 species of
cone snails, which are carnivorous marine gastropods
that use venom for defence, competition, and predation [18.97–100]. Their venoms, termed conotoxins or
conopeptides, have been extensively studied due to the
number and diversity of the neuropharmacologically
active peptides they contain (50 to >1000 per species),
which specifically target a broad range of therapeutically relevant receptors and ion channels [18.97, 99,
100]. Indeed, the drug Ziconotide (Prialt), derived from
the venom of Conus magus, has been approved for
treating severe chronic pain [18.101, 102]. A number
of proteomic studies have been conducted to characterize the venom gland peptidome and proteome of
cone snails in order to increase our understanding of
conotoxin biosynthesis, maturation, secretion, and interspecies variation. A total of 161 and 157 proteins and
protein isoforms were identified in the venom glands
of Conus novaehollandiae and Conus victoriae, respectively, with a large fraction of the proteins found to
function in protein/peptide translation, folding, and protection events [18.99].
In another study, Tayo et al. [18.97] used an LTQOrbitrap tandem mass spectrometer to investigate the
venom proteome in the proximal, central, and distal
portions of the tubular venom duct of Conus textile
in order to elucidate the overall envenomation strategy
employed by Conus species. 31 conotoxin sequences
and 25 post-translational modification variants were
identified following searches using SEQUEST (tandem mass spectrometry data analysis program used for
protein identification) and ProLuCID (name of a tandem mass spectrabased protein identification program)
for unmodified and post-translationally modified peptides, respectively. The study found both qualitative
and quantitative differences in the conotoxin components sampled from the different sections of the