Lectures L7.1 Session 7. Protein Transport and Assembly

Session 7. Protein Transport and Assembly
Lectures
L7.1
Chaperones for folding, assembly
and maintenance of Rubisco
Rca has a wider central pore that might interact with a surface loop of Rubisco. The end result in both cases is the
conformational destabilization of the active center and the
release of the inhibitory sugar. Thus, it appears that organisms with green and red-type Rubiscos have evolved different mechanisms to reactivate the inhibited enzyme.
Oliver Mueller-Cajar1, Matthias Stotz1, Susanne
Ciniawsky2, Petra Wendler2, F. Ulrich Hartl1,
Andreas Bracher1, Manajit Hayer-Hartl1
1Department of Cellular Biochemistry, Max Planck Institute of
Biochemistry, Martinsried, Germany; 2Gene Center Munich,
Department of Biochemistry, LMU, Munich, Germany
e-mail: Manajit Hayer-Hartl <[email protected]>
Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for the fixation of atmospheric CO2
in photosynthesis. Rubisco is not only the most abundant
protein in nature but also one with a remarkably high chaperone requirement for folding, assembly and maintenance.
The major form of Rubisco (form I) is hexadecameric,
consisting of 8 large (RbcL) and 8 small (RbcS) subunits.
The form I Rubiscos are phylogenetically divided into a
green branch, present in cyanobacteria, green algae and
plants, and a red branch, present mainly in photosynthetic
bacteria, red algae and phytoplankton. The red-type Rubiscos are responsible for most oceanic CO2 uptake and many
are able to better distinguish between CO2 and O2 than
their plant counterparts.
Recently, we showed that RbcL subunit folding by the
GroEL/ES chaperonin is tightly coupled with assembly
mediated by the assembly chaperone RbcX. Specifically,
RbcX captures the flexible C-terminal segment of one
RbcL subunit in its central binding cleft and binds another
subunit via its peripheral binding region. Upon binding of
RbcS subunits, the RbcL8RbcX8 complex undergoes an allosteric structural change that results in the dissociation of
RbcX and formation of stable Rubisco holoenzyme. Before the assembled Rubisco can proceed with photosynthesis, its enzymatic center has to be activated.
To acquire and maintain catalytic activity, Rubisco must
first be carbamylated by a non-substrate CO2 molecule at
the active-site lysine and then bind a Mg2+ ion as cofactor. Premature binding of the substrate ribulose 1,5-biphosphate (RuBP) to uncarbamylated Rubisco results in
an inactive complex. Reactivation is catalyzed by a AAA+
(ATPases associated with various cellular activities) protein
called Rubisco activase (Rca), which releases the inhibitory
RuBP in an ATP-dependent manner.
Recently, we solved the crystal structures of green plant
Rca from tobacco and the red-type Rca from the proteobacterium Rhodobacter sphaeroides. Negative stain EM and
biochemical analysis showed that ATP and RuBP must
bind to convert the Rhodobacter activase, called CbbX, into
functionally active, hexameric rings. Tobacco Rca, on the
other hand, forms functional hexamers in the absence of
ATP and has no binding site for RuBP. Furthermore, the
CbbX ATPase is strongly stimulated upon interaction with
Rubisco, while Tobacco Rca is constitutively ATPase active.
Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco
large subunit into the narrow hexamer pore, while Tobacco
The First Polish-German Biochemical Societies Joint Meeting, 2012
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Session 7. Protein Transport and Assembly
L7.2
L7.3
Chaperone proteins in control of protein
aggregation and disaggregation
Cell polarity in myelinating glia: from
membrane flow to diffusion barriers
Szymon Ziętkiewicz, Natalia Litwińczuk Alicja Sobczak,
Agnieszka Jurczyk, Elżbieta Chruściel, Krzysztof Liberek
Mikael Simons
Department of Molecular and Cellular Biology, Faculty of
Biotechnology, University of Gdańsk, Gdańsk
e-mail: Krzysztof Liberek <[email protected]>
When a cell is challenged by temperatures above the physiological level, it launches a heat shock response, increasing
the synthesis of several chaperone proteins to adapt to the
stressful conditions. These heat stress conditions also cause
intracellular protein aggregation. Elimination of aggregates can be achieved by solubilization of aggregates and
either refolding of the liberated polypeptides or their proteolysis. The solubilization and refolding of polypeptides
trapped in these aggregates depend on the collaborative action of chaperones from different families. These include
small heat shock proteins (sHsps), the Hsp70 chaperone
and its cochaperones, and the Hsp100 disaggregases. It has
been proposed that sHsps associate with the aggregating
polypeptides, thus changing their biochemical properties so
that the subsequent Hsp100-Hsp70 mediated disaggregation process becomes much more efficient. The disaggregation process depends on the common mechanism of
substrate threading through the central channel of Hsp100
chaperone. Hsp100 chaperones consist of an N-terminal
domain, mobile M-domain and two ATP-binding domains
(AAA domains) essential for hexamerization and chaperone function. Here, we propose the presence of a novel
structural aspect of disaggregating Hsp100 chaperones
important for specificity and efficiency of disaggregation reaction. Our results suggest that network of ionic
interactions between the first ATPase domain and mobile
M-domain controls the activity of Hsp100 disaggregase.
Disruption of these ionic interactions in Hsp100 disaggregases results in unexpected changes of their biochemical
properties. These included an increase in ATPase activity, a
significant increase in the rate of in vitro substrate renaturation and partial independence from Hsp70 chaperone in
disaggregation. Paradoxically, the increased activities result
in serious growth impediments of yeast and bacterial cells
instead of improvement of their thermotolerance. Our
results suggest that toxic activity is due to ability of mutated disaggregases to unfold, independently from Hsp70,
natively folded proteins.
Max Planck Institute of Experimental Medicine, Department of
Neurology, University of Göttingen, Germany
e-mail: Mikael Simons <[email protected]>
Myelin is a multilamellar membrane which when wrapped
around the axons in a spiral manner ensures fast nerve conduction. Lipids comprise more than 70% of the dry weight
of myelin, a proportion much higher than in other cell
membranes. How oligodendrocytes generate compacted
myelin stacks with a unique molecular composition is unknown. As visualization of membrane trafficking in these
tightly packed, nanometre scaled layers is an extremely
challenging task, we used cultured oligodendrocytes that
establish large, flat 2-dimensional sheets as a model system.
Using this system, we find that oligodendrocytes generate a
barrier that functions as a physical filter to form lipid-rich
myelin membrane sheets. MBP forms this molecular sieve
and restricts the diffusion of proteins with large cytosolic
domains into the sheets. This mechanism is essential for
the generation of the anisotropic membrane organization
of oligodendrocytes and key for the synthesis of compacted, lipid-rich myelin membrane stacks.
47th Congress of the Polish Biochemical Society, 2012
Session 7. Protein Transport and Assembly177
Oral presentations
O7.2
O7.1
Competition between proteasomal
degradation and protein import
in the biogenesis of mitochondrial
intermembrane space proteins
Pharmacochaperoning of the ERretained A1-adenosine receptor
Justyna Kusek, Christian W. Gruber, Christian
Nanoff, Michael Freissmuth
Piotr Bragoszewski, Agnieszka Gornicka, Malgorzata
E. Sztolsztener, Agnieszka Chacinska
Centre for Physiology and Pharmacology, Institute of Pharmacology,
Medical University of Vienna, Vienna, Austria
International Institute of Molecular and Cell Biology in
Warsaw, Laboratory of Mitochondrial Biogenesis, Warsaw, Poland
e-mail: Justyna Kusek <[email protected]>
Background: The A1-adenosine receptor is a member of
the rhodopsin-related subfamily of GPCRs. Point-mutations in the conserved NPxxY(x)5,6F motif at the junction
of helix seven and the C-terminus disrupt surface targeting
of the receptor and result in its intracellular retention. This
trafficking arrest can be overcome by addition of receptor
ligands (pharmacochaperoning) that stabilize the receptor
fold and thus promote surface expression. The mutants
serve as a tool to explore a ramification of the retaliatory
metabolite complex: hypoxia leads to intracellular accumulation of adenosine — by breakdown of ATP and by inhibition of adenosine kinase. Intracellular and extracellular
adenosine levels are in equilibrium because of the action
of the equilibrative nucleoside transporters. Extracellular
adenosine dampens cellular metabolism by acting on inhibitory A1-adenosine receptors and thus counteracts the
impact of hypoxia. If adenosine also pharmacochaperoned
A1-adenosine receptors during hypoxia, it would enhance
its effectiveness as a protective agent.
Materials and Methods: We created cell lines that stably
expressed A1-receptor-Y288A with either a C-terminal YFP
or an N-terminal FLAG-epitope fused to a streptactin peptide. These cell lines were incubated with a combination
of inhibitors to test whether manipulations of intracellular
adenosine levels increased the accumulation of the receptor. The A1-antagonist DPCPX was used as an internal control. In order to mimic hypoxia, the cells were incubated for
24 hours under 5% O2 content conditions. The radioligand
binding and western blotting was performed to determine
the level of mature, binding competent receptors.
Results: The inhibition of adenosine kinase, adenosine
deaminase and equilibrative nucleoside transporters enhanced the accumulation of binding competent receptors.
The action of these inhibitors was as effective as DPCPX
in pharmacochaperoning the receptor. The receptors acquired a mature glycosylation status indicative of export
from the ER and delivery to the Golgi and they reached the
cell surface. Moreover, the action of the enzyme inhibitor
cocktail could also be elicited by hypoxia.
Conclusions: Accumulation of intracellular adenosine
elicits a pharmacochaperoning effect. Accordingly, the retaliatory metabolite concept may be extended to include
a pharmaco-chaperoning (“physiochaperoning”) action of
adenosine. e-mail: Piotr Bragoszewski <[email protected]>
Nearly all mitochondrial proteins are synthesized in the cytosol and translocated into the organelle. Many of them
have signal presequences that are proteolytically removed
by specific peptidases upon their selective import into one
of mitochondrial compartments: the matrix, the inner
membrane or the intermembrane space (IMS). Additionally
various types of non-cleavable targeting and sorting signals
exist. The diversity of signals is matched by the specific
import pathways.
Many IMS proteins share a coiled coil-helix-coiled coil-helix (CHCH) domain, stabilized by disulfide bonds formed
within the conserved cysteine motifs. Upon protein synthesis and release to the cytoplasm, the cysteine residues
of the IMS proteins stay reduced. The IMS proteins are
transported across the outer mitochondrial membrane via
the TOM complex, a main entry gate for all mitochondrial
precursor proteins. The protein precursors destined to the
IMS are recognized by the mitochondrial import and assembly machinery MIA. Subsequently, the oxidative folding catalyzed by MIA is required to trap them in the IMS.
In case of oxidative folding restrictions, for example in
the mutants of the essential MIA components, Mia40 and
Erv1, the IMS proteins accumulate neither in mitochondria
nor in the cytosol, suggesting their efficient degradation.
We present the results of systematic analyses undertaken
by us to determine degradation pathways that are involved
in cellular homeostasis of the IMS proteins.
The First Polish-German Biochemical Societies Joint Meeting, 2012
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Session 7. Protein Transport and Assembly
Posters
P7.2
P7.1
Fluorescent analogues of SFTI-1 —
design, synthesis and biological studies
Mimicking a SURF allele reveals uncoupling
of cytochrome c oxidase assembly from
translational regulation in yeast
Bettina Bareth1, Robert Reinhold1, Martina Balleininger1,
Mirjam Wissel1, Peter Rehling1, 2, David U. Mick1,3
1Department of Biochemistry II, University of Göttingen, Germany;
2Max-Planck-Institut für Biophysikalische Chemie Göttingen, Germany;
3Institute of Biochemistry and Molecular Biology, Freiburg, Germany
e-mail: Bettina Bareth <[email protected]>
Mitochondrial dysfunction in humans is often caused by
defects in the assembly process of the respiratory chain,
leading to severe multisystem disorders. Leigh Syndrome,
a neurodegenerative disorder of infancy, characterized by
necrotic lesions in the brain, can be caused by mutations
of mitochondrial or nuclear DNA. Nuclear gene mutations
leading to Leigh Syndrome most frequently affect SURF1.
SURF1 is a highly conserved assembly factor of the cytochrome c oxidase (COX), the terminal enzyme complex
of the respiratory chain. The molecular function of SURF1
is still not completely understood but has been extensively
studied in its yeast homolog, Shy1. This protein is involved
in the maturation of early assembly intermediate complexes of COX, the accumulation of which negatively regulate
translation of the central subunit Cox1. We have analyzed
missense mutations that affect highly conserved amino acids, identified in Leigh Syndrome patients. We show that
G124 mutations lead to SURF1 precursor proteins that are
effectively imported into mitochondria but undergo rapid
turnover. In contrast, an Y274Dexchange has no effect on
import or stability of SURF1 but leads to impaired function of the protein, which accumulates in a 200 kDa COX
assembly intermediate complex. In yeast, Shy1Y344D does
not lead to an accumulation of COX assembly intermediates, that link to feedback regulation of Cox1 expression.
Instead, Cox1 expression is unaltered and COX assembly is
impaired at later steps.
These data suggest uncoupling of Cox1 expression from
the assembly of the cytochrome c oxidase when Shy1Y344 is
mutated and provide evidence for the dual molecular role
of Shy1. Magdalena Filipowicz1, Anna Łęgowska1, Magdalena
Wysocka1, Adam Lesner1, Adam Sieradzan1, Michał Pikuła2,
Piotr Trzonkowski2, Katarzyna Guzow1, Krzysztof Rolka1
1Faculty
of Chemistry, University of Gdańsk, Gdańsk, Poland; 2 Clinical
Immunology Division, Gdańsk Medical University, Gdańsk, Poland
e-mail: Magdalena Filipowicz <[email protected]>
Trypsin inhibitor SFTI-1 isolated from sunflower seeds
has become one of the most studied peptidic inhibitors of
serine proteases [1]. It is a circular peptide which consists
of 14 amino acid residues with head-to-tail cyclization. Its
structure is also stabilized by a disulfide bridge. SFTI-1 was
found to be homologous to the members of a much bigger
family (BBI), but it is a monofunctional inhibitor. It can
inhibit activity of trypsin, chymotrypsin, human neutrophil
elastase and catepsin G [2, 3]. The reactive site P1-P1’ of the
SFTI-1 inhibitor is located between residues Lys5-Ser6 [1].
These structural features in addition to its strong inhibitory activity make this molecule an attractive template for
designing a new therapeutic agents.
It is known that peptides are not ideal therapeutic agents
because of their limited cell permeability. The aim of this
study was the synthesis of four SFTI-1 analogues with the
attached polyethylene glycol (PEG) moiety at their N-terminus to increase their solubility and cell membrane penetration. The fluorescent reporter groups were also incorporated into the analogues in order to investigate if they
can cross the cell membrane.
The obtained peptidomimetics were used as probes for detection of enzyme – inhibitor complex, which was achieved
using gel filtration chromatography. The fluorescent SFTI1 analogues were subjected to cell penetration assay using
multiple cell lines (HeLa cells and human fibroblast cell
line (46BR.1N)). For all the obtained peptidomimetics, we
determined the association constants with cognate proteinases. References
Luckett RS, Garcia JJ, Barker AV (1999) J Mol Biol 290: 525-533.
Łęgowska A, Dębowski D, Lesner A (2009) Bioorg Med Chem 17: 3302.
Łęgowska A, Dębowski D, Łukajtis R (2010) Bioorg Med Chem 18: 8188.
Acknowledgemets
This work was supported by the project contributing to the development
of young scientist and PhD students under grant No. 538-8290-1039-12.
47th Congress of the Polish Biochemical Society, 2012
Session 7. Protein Transport and Assembly179
P7.3
P7.4
Blood-brain barrier & ethanol:
last point of view
Studies on subcellular localization
of the Germ-cell expressed protein
from Drosophila melanogaster
Nina Grankovskaya1, Mihail Kurbat2
1Department
of General Chemistry, Belarus; 2Department of Biological
Chemistry, Grodno State Medical University, Grodno, Belarus e-mail: Nina Grankovskaya <[email protected]>
The central nervous system (CNS) is a perfectly regulated
environment with conditions far different from those in
the rest of the organism. The blood-brain barrier (BBB) is
the frontier that isolates brain tissues from the substances
circulating in the blood vascular system. One of the major
functions of the BBB is the regulation of the transport of
nutrients and other molecules into and out of the brain.
Polar organization accounts for transport protein localization in brain endothelial cells (ECs) and mediates amino
acid homeostasis in the brain. At least three Na+-dependent excitatory amino acid transporters and one Na+-dependent system transporting glutamine exist at the abluminal side, where as facilitative transporters for glutamate
and glutamine are present only on the luminal side. Such
organization restricts glutamate penetration to the brain. In
addition, there are two facilitative neutral amino acid transporters present at both membranes of ECs. These carrier
sensure the delivery of vital amino acids to the brain.
CNS is a key target organ for the various psychoactive
substances which influence conducts to organic defeat of
structures of a brain and violation of its functional activity. One of such connections is ethanol which thanks to
the physical and chemical properties freely gets through
various biological membranes. Arrived in a stomach and blood ethanol easily gets into CNS where distributed very
non-uniformly. At intoxication development by one of the
first the structures which are a part of BBB are exposed to
influence of ethanol. It is known that ethanol changes permeability of BBB in relation to other substances, including
alien, harmful and toxic.
It is established ethanol affects a brain indirectly, through
its most active metabolite acetaldehyde (AA). Realization of the central action is carried out by AA which is
formed directly in a brain, as a result of ethanol oxidation
by the fabric of a brain. A metabolic barrier to AA as a
part of BBB is aldehyde dehydrogenase of astrocytes. It
is possible to assume that violation of actually barrier and
transport BBB functions as a result of toxic action of the
chemical active AA is a result of damage of membrane
structures of astrocytes and ECs.
Thus, change of permeability of BBB under the influence
of ethanol for various amino acids and other neuroactive
substances, possibly leads to violations of their contents
in a brain. All of it is needing complex scientific research.
Beata Greb-Markiewicz1, Jerzy Dobrucki2, Andrzej Ożyhar1
1Wrocław University of Technology, Faculty of Chemistry, Departament
of Biochemistry, Wrocław, Poland; 2Jagiellonian University, Faculty of
Biochemistry, Biophysics and Biotechnology, Kraków, Poland
e-mail: Beata Greb-Markiewicz <[email protected]>
Opposite to the complexity of vertebrate hormone signaling pathways, D. melanogaster has two known physiologically
active hormones: 20-hydroxyecdysone (20E) and Juvenile
Hormone (JH). Although JH plays important developmental and physiological roles, its receptor for long time
remained undiscovered. The most likely JH receptor candidate is Methoprene-tolerant (Met) which was first identified from D. melanogaster. Germ-cell expressed (Gce) is
the paralog of Met with high homology in the conserved
domains but unknown function (Godlewski et al., 2006;
Abdou et al., 2011; Bernardo & Dubrovsky, 2012). Both
Met and Gce belong to the bHLH-PAS transcription factor family. Functional activity of the bHLH-PAS proteins
is connected with differential usage of the nuclear localization (NLS) and nuclear export (NES) signals. Previously,
we have characterised sequences responsible for Met subcellular localization (Greb-Markiewicz et al., 2011). Presently, we have decided to perform a set of experiments to
identify regions of Gce responsible for its cellular distribution. We have used pEYFP-C1 vector containing enhanced
yellow fluorescence protein for cloning full lenght and fragmented Gce. In the next step, vectors were used for COS-7,
CHO-K1 and HEK293 cells transfection. Finally, localization of expressed fusion proteins was visualised with Confocal Microscopy System. Here we present results of our
experiments.
References
Abdou et al. (2011) Insect Biochem Mol Biol 41: 938-945.
Bernardo TJ, Dubrovsky EB (2012) Insects 3: 324-338.
Greb-Markiewicz B et al. (2011) Mol Cell Endocrinol 345: 16-26.
Godlewski J et al. (2006) Biochem Biophys Res Communi 342: 1305-1311.
The First Polish-German Biochemical Societies Joint Meeting, 2012
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Session 7. Protein Transport and Assembly
P7.5
Grzonka Z et al. (2001) Act Biol Pol 48: 1-207.
Janowski R et al. (2001) Nat Struct Biol 8: 316-320.
Studies of steric zipper motif in
human cystatin C sequence
Acknowledgements
Work supported by grants: DS/8440-4-0172-12 and for Young Scientists:
538-8440-1041-12.
Emilia Iłowska1, Jakub Barciszewski2, Mariusz
Jaskólski2, Aneta Szymańska1 1Department of Medicinal Chemistry, Faculty of Chemistry, University
of Gdańsk, Gdańsk, Poland; 2Center for Biocrystalographic Research,
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań,
Poland
e-mail: Emilia Ilowska <[email protected]>
Research carried out on fibrilization process leads to some
new hypotheses concerning the mechanism of this process,
as well as the factors responsible for triggering and progress of fibril formation by a particular protein. In addition
to external factors, under consideration there are also taken
the intermolecular determinants like the presence of a specific amino acid sequences and/or structural motives, especially in the protein parts exposed to the environment. One
of the currently investigated hypotheses is the “steric zipper” motif, based on the assumption, that in the protein sequence there are short fragments with very high propensity
for formation of the β-stand conformation and subsequent
strong self-association. The propensity of a given sequence
to fulfill the requirements for effective steric zipper can be
calculated theoretically using 3D profile method elaborated
by Eisenberg and coworkers [1]. The basic parameter is the
value of potential energy, calculated by Rosetta algorithm.
The threshold for fibril-forming peptides was established
to be –23 kcal/mol. Even though theoretically the presence
steric zipper sequences was predicted for many proteins,
experimentally it was confirmed only for a few amyloidogenic proteins. The group of Eisenberg isolated and crystallized fibril-forming segment with NNQQNY sequence
from the yeast prion protein Sup35 [2]. Another experiment was carried out for RNase A. In this protein there
are segments with high fibrillization propensity. However,
when the amino acid residues in one of them were rearranged, the propensity for fibril formation was lost. Results
of this experiment suggest strong sequence and weaker
composition dependence of the amyloid-forming peptide
segments [2, 3].
Human cystatin C is a low molecular mass protein. It is
composed of 120 amino acid residues. It has 5 anti-parallel
β-stands and one α-helix. At physiological conditions is a
monomeric protein but under crystallization conditions
forms dimmer [4]. The first three-dimensional structure of
monomeric form of cystatin was determined for chicken
cystatin by X-ray crystallography.
The conformation of hCC was proposed to be very similar to chicken cystatin C, and mutants: V57N [5], V57D, V57P,
V57G (not published) and stab-1 with new disulfide bonds
((L47C)–(G69C)) [6]. The mechanism of the dimerization
of hCC was studied in depth and was shown to base on the
three dimensional domain swapping mechanism [7].
The 3D profile method and calculated Rosetta algorithm
values for hCC show that in this protein there are two fragments, for which high fibrillization propensity can be expected: fragment of loop L1 (Ala52-Asp65) and C-terminal
part of the molecule between Arg93 and Cys117. In this
work results of preliminary studies of nine peptides from
the region of loop L1, fulfilling potential conditions of
steric zipper, will be presented.
References
1. Goldschmidt L et al. (2010) Proc Natl Acad USA 107: 3487-922.
Sawaya MR et al. (2007) Nature 447: 453-73.
Nelson R et al. (2005) Nature 435: 773-84.
Grubb A (2000) Adv Clin Chem 35: 63-695.
Orlikowska M et al. (2011) J Struct Biol 173: 406-136.
47th Congress of the Polish Biochemical Society, 2012
Session 7. Protein Transport and Assembly181
P7.6
P7.7
Bacillus subtilis SepF binds to
the C-terminus of FtsZ
Pilicides effectively hinder chaperone-usher
interactions in all Gram-negative bacteria
Ewa Król1, Sebastiaan van Kessel1, Laura S. van Bezouwen2,
Neeraj Kumar1, Egbert J. Boekema2, Dirk-Jan Scheffers1
Justyna Pilipczuk1,Beata Zalewska-Piątek1,
Krystyna Dzierzbicka2, Sławomir Makowiec2,
Anna Cyranka-Czaja3, Rafał Piątek1
1Groningen Biomolecular Sciences and Biotechnology Institute,
Department of Molecular Microbiology, University of Groningen, the
Netherlands; 2Groningen Biomolecular Sciences and Biotechnology
Institute, Department of Electron Microscopy, University of Groningen,
the Netherlands
e-mail: Ewa Krol <[email protected]>
Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site
of cell division. Formation of the complex starts with the
polymerization of the tubulin-like protein FtsZ into a ring
at midcell. The role of the Z-ring in cell division is critical,
thus it is controlled by other proteins to ensure division at
the correct position and at the right time. Several proteins
directly regulate FtsZ assembly or disassembly by binding
to different regions on FtsZ. Some of these proteins, like
FtsA or ZipA bind to the C-terminus of FtsZ in E. coli.
For B. subtilis not many proteins were found to bind to this
region of FtsZ.
We used a novel pull-down strategy to identify proteins
which interact with the C-terminus of B. subtilis FtsZ. We
fused the C-terminal 69 amino acids of B. subtilis FtsZ to
the HaloTag affinity tag and run lysates of E. coli overexpressing several B. subtilis cell division proteins over the
immobilized C-terminus on beads. Using this method we
found SepF as a binding partner for the C-terminus. We
confirmed that this interaction is highly specific. In a reverse pull-down SepF binds to full-length FtsZ but not to
a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. Using Electron Microscopy we show that the FtsZ C-terminus is required for the
formation of tubules from FtsZ polymers by SepF rings.
We performed an Alanine-scan of the C-terminus to identify residues critical for binding to SepF. Our results show
that two highly conserved residues P372 and F374 proved
to be critical. However, mutation of other residues on the
C-terminus also influences binding strength suggesting that
the overall structure of the C-terminus rather than single
residues plays a role in FtsZ binding to regulatory proteins.
1Department of Microbiology, 2Department of Organic Chemistry,
Gdańsk University of Technology, Gdańsk, Poland; 3Faculty of
Biotechnology, University of Wroclaw, Wrocław, Poland
e-mail: Justyna Pilipczuk <[email protected]>
The chaperone-usher pathway of adhesive structures biogenesis is highly conserved in all Gram-negative bacteria.
Pili are polymers of thousands protein subunits that possess conserved immunoglobuline-like structure denoted
by lack of the seventh G strand. The effect of this structural defect is a hydrophobic acceptor cleft. The folding
of protein subunits is strictly dependent on the action of
specific periplasmic chaperone protein that complements
the defective structure of a subunit by donating a specific
donor strand. Stable chaperone-subunit complex migrates
to the usher protein located in the outer membrane, where
the process of protein subunits polymerization occurs. The
crucial role in this mechanism is performed by chaperone
the structure of which is highly conserved.
The structural and functional conservation of this mechanism renders it a good potential target for development
of a new class of antibacterial agents. Pilicides originally
proposed by Svensson et al. in 2001 are a derivatives of a
dihydrothiazolo ring-fused 2-pyridone scaffold that block
formation of the adhesive organelles by impairing with the
step of interaction between chaperone-subunit complex
and usher protein. The anti-bacterial activity of pilicides as
specific blockers of the chaperone-usher pathway has been
confirmed only in the case of E. coli producing type 1 and
P pili that represent the FGS type organelles. In our works
we addressed a crucial question denoting spectrum of the
anti-bacterial activity of pilicides: Are pilicides the potential anti-bacterial agents that target bacterial virulence in all
pathogenic Gram-negative bacteria encoding chaperoneusher organelles of both FGL and FGS type? As a model
of the FGL adhesive structures we used the uropathogenic
E. coli strains producing Dr fimbriae. Biological evaluation based on the whole-cell assays showed that the E. coli
Dr+ cultivated in the presence of pilicides possessed the
amount of Dr fimbriae reduced to 85 %. This results in the
high inhibition of bacteria adherence propensity to human
cells. To prove that the observed inhibition of Dr fimbriae
assembly is a consequence of pilicide impairing with the
chaperone-usher pathway we measured the relative affinity
of pilicides to the DraB protein using the surface plasmon
resonance technique. Based on the conservation of the
FGL type assembly machinery we conclude that the pilicides should be effective inhibitors of adhesive organelles
biogenesis of this type.
The First Polish-German Biochemical Societies Joint Meeting, 2012
182
Session 7. Protein Transport and Assembly
P7.8
P7.9
Activity of ABC and MDR transporters
of Candida albicans measured
by fluorescence method
Peptides as a new ingredients
in cosmetic products
Joanna Szczepaniak, Marcin Łukaszewicz, Anna Krasowska
Department of Biotransformation, Faculty of Biotechnology, University
of Wroclaw, Wrocław, Poland
e-mail: Joanna Szczepaniak <[email protected]>
Beata Łubkowska1, Beata Grobelna2, Zbigniew Maćkiewicz1
1Department of Organic Synthesis, Laboratory of Polypeptides
Chemistry, 2Department of Analytical Chemistry, Laboratory of
Cosmetic Chemistry, Faculty of Chemistry, University of Gdansk,
Gdańsk, Poland
e-mail: Beata Łubkowska <[email protected]>
Candida albicans is the most common fungal opportunistic
pathogen which colonize mucous membranes resulting in
superficial, though often persistent oral or vaginal candidiasis. Unfortunately, C. albicans is shown to gain different
mechanisms of resistance to commonly used antifungals,
such as azoles. One of the main mechanism of resistance
is overexpression and activity of MDR (multidrug resistance) and ABC (ATP-binding cassettes) transporters which
actively export xenobiotics out of the cell.
The aim of this study was to test different kinds of compounds, including plant extracts, known antifungal drugs
and novel biosurfactants, to find inhibitors of the activity
of MDR and ABC export pumps. It was found that fluconazole, drug commonly used to treat C. albicans infection, is
exported by Cdr1 and Cdr2 exporters. Moreover group of
C. albicans strains with deletions in genes coding MDR and
ABC transporters were used to monitor activity of these
pumps in real time in different environments by measuring
uptake of fluorescent dye 3,3′-dipropylthiacarbocyanine
iodide (diS-C3(3)) into the cell. It was observed that this
fluorescent probe is exported mainly by Cdr1 and Cdr2 but
not Mdr1 transporter and there is significant difference in
efflux of dye between wild-type strains and strains with
pump deletions. Additionally positive correlation between
glucose levels during culture growth or fluorescence measurement and diS-C3(3) export was found.
The technology of cosmetic peptides has been developed
by synthesis of new fragments of peptides, which imitate
sequences occurring in proteins α-collagen and elastin.
Peptides are used in cosmetic products as small molecules
consisting of 6–7 amino acids, but there are also exceptions in the form of 8–10 amino acids, and sometimes even
20 amino acids. The sequence of amino acid has a direct
impact on the size and nature of peptides [1]. Moreover,
it can be observed which part of the molecule has been
responsible for its biological activity. This makes it possible
to create smaller and cheaper compounds with the same or
similar effect [2].
The main task of moisturizing cosmetics, including the
hydrogel masks investigated, is to improve the degree of
hydration of the skin, as well as its appearance [3]. Specific
peptides were designed with the purpose of improving the
effects of cosmetics on skin. When peptides get to the appropriate place in the skin, it can improve its appearance.
Besides, peptides with a corresponding sequence added to
the formula of hydrogel mask can reduce the amount of
preservatives needed in the general procedure. Normally,
peptides are used in many skin care products to improve
the force of moisturizing cosmetics [4].
Moisturizing cosmetics, such as hydrogel masks are very
popular skincare products. They have a very simple composition, are easy to use and are not expensive. In this experiment the formula of cosmetic product in question was
improved by inclusion of a peptide which was synthesized
in the Polypeptides Laboratory. Peptides as active ingredients in cosmetics showed the ability to transport ions,
decrease facial muscle contraction and stimulate human
skin fibroblasts. We attempted the synthesis of signal peptides because of the effects they have on the skin surface.
A series of skin hydration measurements using a model of
pig skin were made. In the end a number of results of the
experiment confirmed beneficial effects of the additional
peptide ingredient in hydrogel masks. Peptides due to their
appropriate sequence and in an adequate concentration
could induce significant effects of moisturizing on skin.
References:
1. Chvapil M, Eckmayer Z (1985) Int J Cosmet Sci 7: 41.
2. Lupo MP (2005) Dermatol Surg 31: 7.
3. Lupo MP, Cole AL (2007) Dermatol Ther 20: 343.
4. Carpino LA, Han GY (1970) J Am Chem Soc 92: 5748.
Acknowledgements
This work was supported by BW/538-8454-1058-12 and DS/8452–4–
0135–12, DS/8210–4–0177–12 and University of Gdansk.
47th Congress of the Polish Biochemical Society, 2012
Session 7. Protein Transport and Assembly183
P7.10
The Franz Diffusion Chamber –
the equipment that measures
permeation through membranes
Beata Łubkowska1, Beata Grobelna2, Zbigniew Maćkiewicz1
1Department of Organic Synthesis, Laboratory of Polypeptides
Chemistry, 2Department of Analytical Chemistry, Laboratory of
Cosmetic Chemistry, Faculty of Chemistry, University of Gdansk,
Gdańsk, Poland
e-mail: Beata Łubkowska <[email protected]>
Nowadays, the penetration behavior of an active ingredient
can be evaluated in vitro, ex vivo, and in vivo. Most of the data
on percutaneous penetration have been gained with in vitro
or ex vivo studies by experiments using a Franz-Diffusion
chamber. The donor (formulation) is separated from the
acceptor (aqueous buffer solution) by an appropriate barrier. For in vitro studies this barrier can consist of an artificial skin construct (ASC). ASC is cultivated from different
cell types and comprises a dermis and a epidermis equivalent [1]. The advantage of ASC is that the properties are
more consistent than in natural skin. However, the barrier
properties of artificial skin are more closely to that of baby
skin. This means it is less restrictive than the skin of adults.
The First Polish-German Biochemical Societies Joint Meeting, 2012