Effects of antitrypsin polymerization of ER structure and function
In 2012, we discovered altered protein mobility in the ER of cells expressing polymerogenic mutants of serpin proteins (Ordonez et al., 2013). We have continued to pursue this as a means to understand the increased sensitivity to ER stress seen in cells accumulating serpin polymers. By defining the effects of polymerised proteins on ER structure, our group demonstrated that Z-alpha1-antitrypsin leads to vesiculation of the ER in association with exclusion of ER shaping proteins including reticulons and atlastins (Dickens et al., 2016). The individual vesicles of functional ER in cells that have accumulated antitrypsin polymers remain functionally connected by the exchange of luminal proteins. This requires SNARE-protein dependent trafficking via the ERGIC. In that study we noted that polymerised Z-alpha1-antitrypsin appears to form a hydrogel within ER inclusions, through which other proteins including chaperones can diffuse. This led us to pursue the biophysical properties of this hydrogel as these are responsible for disruption of ER structure. To this end we developed new technologies including ROVI (rotor based organelle viscosity imaging) (Chambers et al., 2018) and ER-resident crowding probes (Holcman et al., 2018). We have used these technologies to understand the antitrypsin phase transition, bridging the gap in knowledge between biochemical changes to the antrypsin protein and effects on hepatocyte function (Chambers et al., 2022).
Chambers, J.E., M. Kubankova, R.G. Huber, I. Lopez-Duarte, E. Avezov, P.J. Bond, S.J. Marciniak*, and M.K. Kuimova*. 2018. An Optical Technique for Mapping Microviscosity Dynamics in Cellular Organelles. ACS Nano. 12:4398-4407. *Joint senior
Chambers JE, Zubkov N, Kubánková M, Nixon-Abell J, Mela I, Abreu S, Schwiening M, Lavarda G, López-Duarte I, Dickens JA, Torres T., Kaminski CF, Holt LJ, Avezov E., Huntington JA, St George Hyslop P, Kuimova MK, Marciniak SJ. (2022). Z-⍺1-antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state. Science Advances [in press].
Dickens, J.A., A. Ordonez, J.E. Chambers, A.J. Beckett, V. Patel, E. Malzer, C.S. Dominicus, J. Bradley, A.A. Peden, I.A. Prior, D.A. Lomas, and S.J. Marciniak. 2016. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-alpha1-antitrypsin. FASEB J. 30:4083-4097.
Holcman, D., P. Parutto, J.E. Chambers, M. Fantham, L.J. Young, S.J. Marciniak, C.F. Kaminski, D. Ron, and E. Avezov. 2018. Single particle trajectories reveal active endoplasmic reticulum luminal flow. Nat Cell Biol. doi: 10.1038/s41556-018-0192-2.
Ordonez, A., E.L. Snapp, L. Tan, E. Miranda, S.J. Marciniak*#, and D.A. Lomas*. 2013. Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency. Hepatology. 57:2049-2060. *Joint senior. #Corresponding
Role of eIF2α phosphorylation in diseases
The effect of ER stress on cell growth and survival is relevant to the pathogenesis of many diseases including cancer. The mechanisms linking ER stress with cell death are complex, but to a large extend involve regulation of new protein synthesis. Understanding these processes has been a major focus of our studies.
In 2013, we identified a relationship between the expression levels of some downstream targets of the ER stress response and the rate of progression of malignant mesothelioma (Dalton et al., 2013). We went on to describe mechanisms by which ER stress signalling, specifically that downstream of eIF2α phosphorylation, affect cell cycle progression and death in mammalian cells (Thomas et al., 2013) and Drosophila (Malzer et al., 2010).
Phosphorylation of eIF2α occurs in response to a variety of stresses in addition to ER stress and so the shared downstream pathway is called the integrated stress response (ISR). For example, in collaboration with Pieter Hiemstra, Leiden University, we showed that the iron sensing eIF2α kinase HRI triggers the ISR during infection with Pseudomonas (van ‘t Wout et al., 2015).
Dalton, L.E., H.J. Clarke, J. Knight, M.H. Lawson, J. Wason, D.A. Lomas, W.J. Howat, R.C. Rintoul, D.M. Rassl, and S.J. Marciniak. 2013. The endoplasmic reticulum stress marker CHOP predicts survival in malignant mesothelioma. Br J Cancer. 108:1340-1347.
Malzer, E., M.L. Daly, A. Moloney, T.J. Sendall, S.E. Thomas, E. Ryder, H.D. Ryoo, D.C. Crowther, D.A. Lomas, and S.J. Marciniak. 2010. Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response. J Cell Sci. 123:2892-2900.
Thomas, S.E., E. Malzer, A. Ordonez, L.E. Dalton, E.F.A. van ‘t Wout, E. Liniker, D.C. Crowther, D.A. Lomas, and S.J. Marciniak. 2013. p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress. J Biol Chem. 288:7606-7617.
van ‘t Wout, E.F., A. van Schadewijk, R. van Boxtel, L.E. Dalton, H.J. Clarke, J. Tommassen, S.J. Marciniak*, and P.S. Hiemstra*. 2015. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells. PLoS pathogens. 11:e1004946. *Joint senior
Mechanisms that regulate eIF2α phosphorylation
In 2004, we discovered that the transcription factor CHOP determines cell survive, at least in part, through induction of a phosphatase named GADD34 (also know as PPP1R15A) (Marciniak et al., 2004). We also discovered the mechanism by with the functional antagonist of GADD34, a kinase named PERK, achieves selective recruitment of their shared substrate eIF2α (Marciniak et al., 2006).
By describing the first invertebrate eIF2α phosphatase (or PPP1R15 enzyme) we discovered a role for the ISR during Drosophila larval development (Malzer et al., 2013). By comparing the protein-binding partners of the mammalian PPP1R15s and the Drosophila homologue, we identified G-actin as a conserved component of the active holoenzyme (Chambers et al., 2015). Moreover, we discovered that actin dynamics regulate the activity of PPP1R15 by affecting G-actin availability and so tune the sensitivity of the ISR. In collaboration with David Ron and Randy Read, we went on to elucidate the molecular mechanism for this regulation.
Chambers, J.E., L.E. Dalton, H.J. Clarke, E. Malzer, C.S. Dominicus, V. Patel, G. Moorhead, D. Ron, and S.J. Marciniak. 2015. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2alpha dephosphorylation. eLife. 4.
Malzer, E., M. Szajewska-Skuta, L.E. Dalton, S.E. Thomas, N. Hu, H. Skaer, D.A. Lomas, D.C. Crowther, and S.J. Marciniak. 2013. Coordinate regulation of eIF2alpha phosphorylation by dPPP1R15 and dGCN2 is required during development. J Cell Sci. 126:1406-1415.
Marciniak, S.J., L. Garcia-Bonilla, J. Hu, H.P. Harding, and D. Ron. 2006. Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK. J Cell Biol. 172:201-209.
Marciniak, S.J., C.Y. Yun, S. Oyadomari, I. Novoa, Y. Zhang, R. Jungreis, K. Nagata, H.P. Harding, and D. Ron. 2004. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev. 18:3066-3077.