Future work

Our work has demonstrated that STING-dependent type I interferon expression is

regulated by the novel proinflammatory mediator TMEM203. The function of

TMEM203 was previous reported to relate to calcium signalling, while our data strongly

support a previously unidentified immune regulatory role. As my project mainly

investigated the role for TMEM203 and STING IFN-I induction, a number of areas

could be explored regarding the additional functions of TMEM203.

One area to expand is the LPS-induced TMEM203 upregulation, initially discovered in

the cDNA functional screen in RAW 264.7 cells. As the project evolved, STING

became the priority area of research, but questions remain as to how TMEM203

expression is upregulated by LPS stimulation and whether TMEM203 acts as the

intermediate platform for crosstalk between LPS/TLR and STING pathways. There is

a lack of strong evidence for STING and TLR signalling communication, although an

intact microbe presents multiple PAMPs that can be recognised by several PRRs. For

example, the bacterium Brucella abortus is able to activate both TLR/MyD88 and RNA

polymerase III/STING pathways [290]. Further experiments could explore the function

of TMEM203 in the context of LPS-induced inflammation and address the mechanistic

pathways of STING regulation. This area is of significant interest to link complex

antimicrobial immune signalling between STING and TLR.

Commercial purified STING ligands (Invitrogen) were used throughout experiments as

the focus was to identify and monitor the interaction between TMEM203 and STING,

and thus the use of complex microorganism was not required. However, human

physiology almost never encounters clean bacterial molecules, but rather as

biologically active organisms that can change and interact with host system. It is yet

unclear whether complex immune stimuli may induce alternative TMEM203 activation

other than pathway via STING. The aspect of TMEM203-dependent chemokine

upregulation by LPS challenge was under-addressed, and questions arise as whether

TMEM203 can switch between signalling routes in response to different immune

challenges. Furthermore, the mechanism underlying re-localisation of TMEM203

induced by LPS was unclear (Chapter 2, Fig. 3A-B). Whether LPS or other bacterial

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ligands could promote TMEM203 translocation to lysosomes and thus potentiates

STING activation will require further investigation.

In Chapter 2, we have extensively investigated the localisation and translocation of

TMEM203 and TMEM203-STING complex to provide reason for their interaction in

immune activation. However, limited by the little cytosolic exposed region in TMEM203,

our investigation on TMEM203 relies solely on the expression of fusion plasmids which

inevitably resulted in the overexpression of this gene. Overexpression of ER proteins

has been implicated in unfolded protein stress response which disturbs the

physiological condition of the organelle, creating an additional variable in the

experiment system [291, 292]. To eliminate the possibility, we have measured the

transcriptional splicing of XBP1 as an indicator of ER stress unfolded protein response

(UPR), and little stress event was detected (Chapter 2, Fig. S4). Despite this,

TMEM203 upregulation could possibly induce calcium perturbation and showed an

intracellular localisation pattern of its post-activated state [132]. A specific anti-

TMEM203 antibody will be required to solve this problem, as well as to quantify the

protein levels in TMEM203 knockdown experiments in human monocyte-derived

macrophages (Chapter 2, Fig. 4A-B). More importantly, the time and spatial regulation

of TMEM203 will be better characterised with antibody labelling which may reveal

differential trafficking strategies to various immune challenges.

Protein complementation assay was frequently used in this project to investigate

protein-protein interaction. This technique shown in Chapter 2 Fig. 6A-C has helped

us to quantify molecular interaction in living cells. Although cells are monitored in a

short 30-minute period under restricted CO

-free condition, we were able to capture

the association of proteins at 2.5 to 5-minute time points. Such assays can be

developed into tools for in vitro drug screen [293, 294]. The advantage of this

experiment was to compensate the unspecific false positive protein interactions reveal

by a protein-only screen test, as live-cell drug screen ensures that proteins are

available in the native cellular environment with all potential influence of natural

inhibitors and scaffolding proteins, and it also assesses the permeability of drugs

across the plasma membrane. Combining this live-cell assay with hi-throughput

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analysis [294] can be both effective and accurate in validating pharmacological

influence on protein association in a real physiological setting. Similar live-cell based

PCA has already been used to screen protein interactions using the reporter of

dihydrofolate reductase (DHFR) to reflect protein interactions by yeast growth under

different chemical conditions [295]. In comparison, our system is superior in rapid

detection of protein interactions via luciferase reporter, and hence it can precisely

distinguish both the strength and duration of interaction.

Although the interaction and functional relevance between STING and TMEM203 has

been established, we have to acknowledge that much is unknown about the activities

of TMEM203 in regulating inflammation. It is still unclear whether TMEM203 is a

stabiliser of STING dimerisation and functional conformation, whether TMEM203 also

couples to additional receptors or ion channels (most likely calcium channels), and

whether TMEM203-STING interaction is a promising therapeutic target for STING

deficiency or over-activation. Although we have analysed the level of TMEM203,

STING and MAVS in T cells isolated from a cohort of treatment naïve patients, it still

remains unclear how these genes contribute to or affected by this pathology, or if there

are other cell types that can better represent the roles of these interferon regulators.

Particularly, patient-derived T cells were activated by PHA (phytohaemagglutinin) prior

to TMEM203 and STING mRNA measurement, and its potentially impact to T cell

characteristics and interferon response should be addressed in future. Although we

have demonstrated the role for TMEM203 and STING in a variety of macrophage

models, it is crucial to ascertain whether they have similar functions in other type I

interferon-producing cells, particularly T cells which largely contribute to SLE [139].

Furthermore, a loss-of-function STING HAQ (R71H-G230A-R293Q) variant has been

identified to show 90% reduction in the ability to activate IFN-β signal in response to

Legionella pneumophila and bacterial STING ligands [296]. The series of mutations

occurs in the transmembrane domain and ligand binding domain of STING which

impairs but not completely abolishes STING’s ability to become activated. Thus, the

HAQ mutant would likely alter STING’s ability to dimerise and interact with TMEM203.

Questions also remain to the potential of single nucleotide polymorphisms (SNPs) in

TMEM203 gene, nevertheless there was none discovered at present, this would be

worth investigating in future.

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Particularly, since STING overactivation can cause autoimmunity and its deficiency

increases infection susceptibility, the challenge for targeting STING therapeutically is

to identify the fine balance for its activation and inactivation. Based on our evidence,

TMEM203 has the potential to be a surrogate for STING intervention because of its

ability to alter but not complete abolish STING signalling. This can ideally prevent

excessively immunodeficiency and renders the host certain ability to exhibit interferon

response. Further studies on the human and mouse STING orthologues can also

inform us how the TMEM203-dependent regulation in these two species will likely be

comparable or distinct in antiviral response. In general, the discovery of TMEM203

has widened our knowledge on inflammatory signalling and has added a substantial

piece of evidence to address the importance of STING-regulated immunity.

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Chapter 4 (Paper 3): STING regulation in response to