Download Structural biology reveals links between Inflammation and Metabolic

FINAL REPORT 2016-03-31
Projekttitel
Structural biology reveals links between Inflammation and Metabolic disease
Projektledare
Associate Professor Karin Lindkvist, Head of Medical Structural biology, Lund University.
Progress
The project proposal had 5 aims (Aim #1 Can superantigens interact with the IL-6 signaling complex and what are
the consequences?, Aim #2 Can superantigens affect insulin function?, Aim #3 What role does glycerol play in
glucose homeostasis?, Aim #4 What determines the substrate specificity and rate of transport of glucose and
glycerol?, Aim #5 Can inhibitors targeting glucose or glycerol transporters and lead to new drugs against cancer?),
although the project was funded by AFA it was only enough to employ one person, while in the proposal it was
stated that two persons are needed to execute the full project in time. However, several parts of the project have
been executed and published, or soon-to-be published.
Results
The research aims of the project were:
1. To characterize the binding properties of superantigens with gp130 through
structural and functional in vitro analyses.
Results: The Superantigen, SEA, was shown to bind to gp130 in the micromolar range using
SPR-analyses (Banke et al., 2014). Moreover, SEA was also shown to activate gp130
signaling pathway and affect the function of adipocytes. These data have been summarized in
an article published in Metabolism in 2014(Banke et al., 2014). Moreover, we have been able
to show that activation of gp130 and binding to it is not solely performed by the superantigen
SEA, instead a whole repertoire of superantigens (SEE, SEH, SED) from S. aureus are also
able to perform this action (Regenthal et al, manuscript in preparation). Moreover, we have
determined the structure of SEA, SEE and SEB together with its other receptor, TCR and
recently published (Rodstrom et al., 2014; Rodstrom et al., 2015).
Gp130 was loaded on the BIAcore chip and superantigens were run over at different
concentrations. All superantigens shown above bind to human gp130.
2. To reveal the effects on glucose/glycerol transporters caused by superantigens in adipocytes
as well as in skeletal muscles.
Results SEA was shown to bind human gp130 (aim 1). Another aim for this project was to
study the effects of SEA in human adipocytes (fat cells) instead of rat adipocytes, as
previously been used. We have during the past year set up collaboration with clinicians in
Malmö to receive human fat tissue. We have seen that SEA can bind to gp130 on the surface
of human adipocytes using microscopy. As seen below, the red stain (superantigen) and the
green stain (gp130) co-localize since they turn yellow when they are overlaid.
Moreover, we have preliminary data supporting that insulin induced glucose uptake is affected
by superantigens, hence the adipocytes become partly insulin resistant when treated with
superantigens, as seen below. These data will be combined in a manuscript in the near future
and published.
3. To determine the trafficking pattern of glycerol transporters and its role in metabolic
disorders.
Results: The aim was determine in detail the trafficking pattern of glycerol transporters in
human adipocytes. We have during the past years investigated the molecular mechanism
controlling AQP7 mobility in human primary adipocytes. AQP7 is the major glycerol efflux
channel in adipocytes causing adipocyte hypertrophy when deleted in mice, and is known to
translocate between subcellular structures upon hormone stimulation. We have discovered
that AQP7 and perilipin 1 form a complex in adipocytes that is released upon stimulation by
catecholamines ex vivo, or by PKA-mediated phosphorylation in vitro. We have also identified
that PKA targets the N-terminus of AQP7. Hence, we can now finally in detail describe the
regulation of glycerol channels in human adipose cells. The manuscript (submitted to
Scientific reports)(Krintel, 2016) provides a unique insight into how glycerol channels are
controlled in adipocytes and paves the way for future design of drugs against human metabolic
pathologies. Figures below show the physical interaction between AQP7 and PLIN1
investigated in vitro (A), and the physical interaction investigated ex vivo in human adipocytes
(B). Briefly, AQP7 was spotted onto a membrane, and incubated with lysate including PLIN1
(PLIN1-lysate), if AQP7 can capture PLIN1, signal will appear. As seen below, a clear signal
is seen from the AQP7 spot, as well as the positive control (PLIN1 lysate) (A). In B, a
proximity ligation assay was used to determine whether AQP7 and PLIN1 are in close
proximity in the cell. Clear red-spots are detected, confirming that AQP7 and PLIN1 are in
close proximity also in the cell. Moreover, C-D show that the complex between AQP7 and
PLIN1 is released upon catecholamine stimulation (isoprenalin). The data below is currently
under review for publication in Scientific reports.
a
c
4. To characterize the structure and transport properties of glucose and glycerol transporters
Results Both the glucose and the glycerol transporters have been successfully expressed
and crystallized. One glycerol transporter has been particularly successful and we have a
data set to 3.9Å resolution. We need to improve it further but are currently working on this.
For the glucose transporters, we have recently started a collaboration with Prof. Nieng Yan
in Beijing (supported by the Swedish Research Council) who recently determined the
structure of both GLUT1 and GLUT3, which was part of this project. We are currently
together aiming and structurally determine the GLUTs together with putative inhibitors
(see next project goal)
5. To design and test putative inhibitors for glucose and glycerol transporters, in particular for
use in tumor therapy.
Results To design and test inhibitors for glucose and glycerol
transporters the major obstacle is to set up a reliable assay. We have
during the past years worked on this, with main focus on the glucose
transporter. Still, the assay is transferable to the glycerol transporters,
as well. The purified proteins (from aim 4) have been inserted into
giant vesicles (artificial vesicles roughly the same size as regular
cells). If the proteins are functional (can import glucose into the
vesicles), the “cells” will light up (see picture to the right). This data
has been publihsed in Chemm. Comm. (Hansen et al., 2015).
Moreover, we have started a collaboration with Prof. Filippo
Minutolo and we are currebtly testing his inhibitors for glucose
transport. As postive control we use the well-known inhibtor
cythochalsin B (CB). As seen below, we have to inhibitors (PGL-13
and PGL-17) that have promsing results. These inhibitors will be
used both for optimization of new inhibitors and also to test for
inhibition of growth of leukemic cancer cellines, focusing on ALL and MLL.
Deviations from the project plan
We have more or less followed the project plan, but since we only received half of the money
requested roughly 75% of the aims have been executed (as seen above). However, we plan to
finalize the last part during the next coming years.
How will the data be disseminated to the public sector
All data that we generate are published in well-known peer-reviewed journals. Moreover, we
write several more popular scientific articles to also reach scientists and Medical Doctors that
are not in the field or working clinically (Lindkvist-Petersson, 2012, 2013, 2016). Finally, the
putative cancer drugs that we are currently investigation will in the future be tested clinically.
References within the project:
Banke, E., Rodstrom, K., Ekelund, M., Dalla-Riva, J., Lagerstedt, J.O., Nilsson, S., Degerman, E.,
Lindkvist-Petersson, K., and Nilson, B. (2014). Superantigen activates the gp130 receptor on
adipocytes resulting in altered adipocyte metabolism. Metabolism: clinical and experimental.
Hansen, J.S., Elbing, K., Thompson, J.R., Malmstadt, N., and Lindkvist-Petersson, K. (2015).
Glucose transport machinery reconstituted in cell models. Chem Commun (Camb) 51, 2316-2319.
Krintel, C., Hansen J.S., Hernebring M., Haataja T.J.K, de Marè S., Wasserstrom S., KosinskaEriksson U., Palmgren M., Holm C., Stenkula K.G., Jones H.A., Lindkvist-Petersson K. (2016).
Perilipin 1 binds to AQP7 in human adipocytes and controls its mobility via PKA mediated
phosphorylation. Under review in Scientific Reports.
Lindkvist-Petersson, K. (2012). Att svälta tumören-möjlig cancerterapi i framtiden. . Akutellt om
Vetenskap och Hälsa.
Lindkvist-Petersson, K. (2013). Grönt te - framtida läkemedel för cancerbehandling? Onkologi i
Sverige.
Lindkvist-Petersson, K. (2016). Kan strukturbiologi avslöja mekanismerna för hur bakterier bidrar
vid utveckling av metabolasjukdomar? . Best Practice: DIABETES/HJÄRT-KÄRLSJUKDOMAR To
be published.
Rodstrom, K.E., Elbing, K., and Lindkvist-Petersson, K. (2014). Structure of the superantigen
staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCRpeptide contacts. J Immunol 193, 1998-2004.
Rodstrom, K.E., Regenthal, P., and Lindkvist-Petersson, K. (2015). Structure of Staphylococcal
Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen
Recognition. PloS one 10, e0131988.