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Nutrition & Metabolism
Cell Signalling & Gene Expression
PHD and Honours Projects, 2017
Prof. Proud’s laboratory studies the signalling pathways by which hormones, growth factors and nutrients
regulate the function of mammalian cells, especially protein metabolism. The proper control of these pathways
plays an important role in cell growth and proliferation, and in neurological processes such as learning and
memory. Defects in their control contributes to tumorigenesis, type 2 diabetes, cardiovascular disorders and
neurodegenerative disease.
A major nutrient-sensitive signalling pathway involves the mammalian target of rapamycin, complex 1,
mTORC1. mTORC1 is activated by hormones and amino acids, and regulates multiple steps in protein
synthesis including the initiation and elongation stages. We are also currently investigating the role of several
protein kinases closely related the mTORC1 pathway, all of which directly impact mRNA translation. As direct
regulators of key protein synthesis factors, these kinases, which include MNK1, MNK2 and eEF2K, play a
central role in cellular metabolism. MNK1 and MNK2 phosphorylate the key translation initiation factor
eIF4E.
Project 1 is available as a PhD or Honours project, while project 2 is available for MPhil / Honours.
1. Oncogenic potential of constitutively active (CA) Rheb (Ras-homolog enriched in brain) mutations.
BACKGROUND:
mTORC1 is a master molecular hub which couples nutrient and growth factor signals to control both key
anabolic and catabolic processes within the cell. Importantly, many reports in recent years have indicated that
cancer cells harbour a number of mutations found in genes related to the mTORC1 signalling pathway, which
results in the over-activation of mTORC1 and hence promoting cancer progression. Classic examples of this
include upstream regulators of mTORC1 such as PTEN (phosphatase and tensin homolog), B-Raf, TSC
(tuberous sclerosis complex) and mTOR itself. Many of these are indeed reported oncogenes, which are
mutated genes that lead to tumour initiation and progression.
Genetic and biochemistry studies have provided evidence
that a small G-protein named Rheb is crucial for mTORC1
activation. Of interest, several mutations in the Rheb gene
have recently been found in tumorous organs/tissues from
cancer patients, including urinary tract, lung, large
intestine, breast, kidney and endometrium. We have
recently found that several of these mutations confer Rheb
resistance towards upstream inhibitory signals and
therefore lead to the formation of a constitutively active
(CA) Rheb and subsequently over-activation of mTORC1.
Notably, a previous study has indicated that CA Rheb
mutants possess great oncogenic properties and would
greatly promote the transformation of normal healthy cells
into cancer cells (Jiang & Vogt, Oncogene, 2008).
AIMS:
In this project, we will explore the oncogenic potential of
these Rheb mutations in transforming normal cells into cancer cells. We will use the state-of-the-art
CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) gene editing technology to introduce
cancer-associated mutations into the chromosomal copies of Rheb genes in NIH3T3 fibroblasts as a model to
generate stable cell lines harbouring these Rheb mutations. The oncogenic potential of these cells will then be
tested by conventional cell transformation assays. We will study the effects of active Rheb mutants on cell
growth, proliferation and cell
cycle, and on protein synthesis. Fig 1. Schematic presentation of the oncogenic signalling pathways involving the RhebWe will also explore how CA mTORC1 which couples hormones and growth factor signals to tumourigenesis.
Rheb mutants affect cell
function by comparing the spectrum of proteins being synthesized in cells bearing CA Rheb and those with
wild-type Rheb, by using the BONCAT (bioorthogonal non-canonical amino acid tagging)-pSILAC (pulsed
stable isotope-labelling with amino acids in cell culture) technique coupled with mass-spectrometry.
Key reference:
Jiang, H. and Vogt, P.K. (2008) Oncogene. 27:5729-5740. Constitutively active Rheb induces oncogenic
transformation.
Wang, Y., Hong, X., Wang, J. Yin, Y., Zhang, Y., Zhou, Y., Piao, H-I., Liang, Z., Li, G., Xu, G., Kwiatkowski,
D.J. and Liu, Y. (2016) Oncogene. In press. Inhibition of MAPK pathway is essential for suppressing RhebY35N driven tumor growth.
2. Systematic analysis of the eIF4E interactome: understanding the distinct functional roles of the three
mammalian eIF4E proteins in translation initiation.
BACKGROUND:
One of the most intriguing findings to arise from the recent availability of genome-wide datasets measuring
the rates of synthesis and turnover of both mRNA and protein is that cellular protein abundance is determined
mainly by controls at the level of translation. Indeed, the majority of variance (~55%) in protein levels is
determined by the rates of translation of their mRNAs. Translational control of protein amount has several
advantages: for example, one, production of a given protein can be quickly activated at the ribosome, without
the need to first transcribe, process and export the template mRNA, and two, a protein’s synthesis can be
restricted to specific subcellular locales, which is essential in polarised cells such as neurons or cells
undergoing directed migration.
While we now understand a great deal about the general mechanisms of translation, our insights into the
broader picture of how both cellular environment and sequence-specific translational regulatory mechanisms
cooperate to control the translational efficiency (TE) of a given mRNA under specific cellular states are poor.
However, it is clear that translation initiation, i.e., the step where ribosomes are recruited onto the mRNA and
locate the start codon, is a key site of regulation. Initiation involves the association of the mRNA with a set of
proteins termed eukaryotic initiation factors, eIFs. There are 3 core eIFs (‘the eIF4F complex’): the mRNA 5′
cap-binding protein eIF4E; the RNA helicase eIF4A; and the scaffold eIF4G, which interacts with eIF4E,
eIF4A and PABP (which in turn also binds the 3′ poly(A) tail of the mRNA). The eIF4F:mRNA assembly –
the classic 'closed-loop' structure – provides a platform onto which the 40S small ribosomal subunit, already
bound to other translation factors and termed the 43S pre-initiation complex [PIC]) docks, putting in motion
the process of translation
Interestingly, while the vast majority of translation initiation research has studied the eIF4E-type protein
eIF4E1, there are two other mammalian 4E isoforms (arising from two other genes); we henceforth refer to
them as 4E1, 4E2 and 4E3 (the 4Es). We recently made the significant discovery that 4E2 may act as the key
mediator of a stress response that achieves selective translational inhibition of mRNAs involved in cellular
growth. As the field lacks even basic experimental data on 4E2 and 4E3, understanding our important and
novel findings requires a research strategy that yields comprehensive data on the mRNA and protein cohorts
of each 4E, and uses the data to build and test models of 4E function.
AIMS:
B
In this project we will probe A
stop
stop
how changes in the cellular
environment
affect
the
individual
functions
and
AUG
AUG
macromolecular interactions of
m7G
4E1, 4E2 and 4E3. In brief, we
prevents
43S PIC joining
plan to investigate, using
43S PIC
joining
m7G
P P P P
human cell lines grown in
culture, how alterations to the
active mTORC1
mTORC1 inhibited
cellular environment that have
been
designed
to
alter Fig. 2 (A) Active mTORC1 phosphorylates 4E-BP, preventing 4E1 binding. (B)
mTORC1 (mammalian target mTORC1 inhibition leads to 4E-BP hypophosphorylation; 4E-BP now binds 4E1
of rapamycin complex 1) and blocks eIF4F.
signalling affect both the mRNA and protein partners of the three 4Es. As mTORC1 activity has been shown
to play a major role in the regulation of translation initiation (partly by mechanisms which are well understood
and partly through unknown means) we plan to identify the individual mRNAs and protein partners of each
4E under each of the five cellular growth conditions. Next, to understand how these cellular environments alter
translation, we will use polysome profiling to determine the TE of individual mRNAs, combined with pulsed,
isotopic labelling of cellular proteins to allow a direct readout of translational activity at the individual protein
level. Finally, by integrating the information we will obtain about the translational activity of individual
mRNAs with our knowledge of each message’s 4E isoform association, across five different cell contexts, we
should gain a number of novel and comprehensive insights into the functional roles of each 4E in translation
initiation which can then be tested by experiment. This project will require contributions from a number of
researchers, and we expect there will be major roles for both PhD and MPhil/Honours students across this
ambitious proposal.
Key references:
Schwanhäusser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W., and Selbach, M.
(2011). Global quantification of mammalian gene expression control. Nature 473, 337-342.
Huo, Y., Iadevaia, V., and Proud, C. G. (2011). Differing effects of rapamycin and mTOR kinase inhibitors
on protein synthesis. Biochem Soc Trans 39, 446-450.
3. Cellular-stress induced activation of non-TFEB regulated lysosomal genes
BACKGROUND:
Imperative to the survival of the mammalian cell is the ability to catabolise macromolecules that are
detrimental to its function, whether they be intrinsically toxic at any level, or otherwise harmless or even
necessary until a threshold has been reached [1]. Lysosomes play a key role in this as they are the sites of
autophagy, a process which also comes into play during nutrient starvation, allowing cells to break down, e.g.,
non-essential proteins to provide amino acid to create new proteins that the cell does need.
Lysosomes are also a key signalling hub being, for example, the location where active mTORC1 (mammalian
target of rapamycin complex 1) resides. mTORC1 integrates nutrient and other signals to control anabolic
processes such as protein synthesis and catabolic ones such as autophagy (and lysosome biogenesis).
The process of lysosomal biogenesis ensures that cells maintain adequate levels of lysosomes. Lysosomal
dysfunction is linked to a range of diseases, such as neurodegenerative disorders, and we therefore need a
better understanding of the control of their production.
Lysosomes are generated at a steady state in normal conditions. However, there is an upregulation in the
expression of lysosomal genes, and a subsequent increase in lysosome numbers and size during cellular insults,
such as an increase in toxic products, or nutrient depletion. Cellular stress-induced transactivation of most
lysosomal genes is mediated by the transcription factor EB (TFEB) [2]. In normal conditions, TFEB is
maintained in the cytoplasm in an inactive form by mTORC1 signalling. However, in conditions of cellular
stress, such as starvation or increased lysosomal storage, TFEB translocates to the nucleus, thus allowing it to
increase the expression of its target genes.
The DNA consensus sequence to which TFEB binds is known as the Coordinated Lysosomal Expression and
Regulation (CLEAR) element. Most lysosomal genes have at least one CLEAR element, and are
transcriptionally upregulated by TFEB [2;3]. However, not all lysosomal genes contain a CLEAR element or
are regulated by mTORC1. Cellular stresses also increase the expression of non-TFEB regulated lysosomal
genes, although the mechanisms by which this occurs remain elusive.
AIMS: this project aims to gain new insights into the control of lysosome biogenesis especially the regulation
of those genes which are not regulated by TFEB. One other pathway by which expression of lysosomal genes
may be regulated involves the translation factor eIF2α which controls the expression of the key transcription
factor, ATF4. Here, in cases of amino acid deprivation or endoplasmic reticulum stress, eIF2α is
phosphorylated, which in turn increases levels of ATF4 protein which, directly or indirectly, increases the
transcription of a subset of genes implicated in autophagy [4]. We will investigate the role of this signalling
pathway in the cellular-stress induced increase in non-TFEB gene transcription. Both eIF2 and ATF4 play
important roles in response to cell stress and in neurodegenerative disorders such as Alzheimer’s Disease.
METHODS: We will employ mammalian cell models to explore the role of the mTORC1 and eIF2/ ATF4
pathways in lysosome biogenesis. Quantitative real-time polymerase chain reaction (qRT-PCR) will be used
to determine the levels of lysosomal genes in response to pharmacological and genetic activators and inhibitors
of the eIF2α/ATF4 pathway. Chromatin immunoprecipitation (ChIP) assays will be utilised to determine which
candidate transcription factors (such as ATF4) bind to specific genes. Finally, reporter assays will be used to
delineate the specific DNA sequence (or response element) to which the transcription factor binds. The host
lab. has extensive expertise in studying these pathways and lysosome biology.
KEY REFERENCES:
1. Ballabio A. 2016. EMBO Mol Med 8: 73-76. 2. Sardiello M, et al. 2009. Science 325: 473-477. 3. Palmieri
M, et al. 2011. Hum Mol Genet 20: 3852-3866. 4. B’chir W, et al. 2013. Nucl Acids Res 41: 7683-7699.
Contact:
SAHMRI
North Terrace
Adelaide SA 5000
Email: [email protected]
Website: www.sahmri.com