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Institute for Research in Biomedicine
Istituto di Ricerca in Biomedicina

Via Vincenzo Vela 6 - CH-6500 Bellinzona
Tel. +41 91 820 0300 - Fax +41 91 820 0302 - info [at] irb [dot] usi [dot] ch

Cellular Responses to Variations in ER Homeostasis and Protein Load

Research area: Controllo della produzione proteica

Group leaders: Maurizio Molinari

Researchers:

Status: In progress

The equilibrium between protein synthesis, export and ERAD is crucial for maintaining ER homeostasis. Different physiological and pathological conditions (e.g. fluctuations in protein synthesis, accumulation of defective gene products, pathogens…) can perturb the ER environment, leading to conditions of ER-stress. Such stresses can lead to the activation of the UPR, adaptive, transcriptional and translational programs that induce the expression of ER resident gene products, increase the ER size and reduce synthesis of cargo protein in order to restore ER homeostasis. The aim of this project is to investigate how cells respond to perturbations of the ER environment upon tunable expression of a selection of model proteins with different physico-chemical features or upon exposure to ER-stress inducing drugs. Experimental data from the lab show that cells respond differently to increasing amount of misfolded ER proteins. While some proteins do not induce an UPR even at high molar concentrations (Figure 1A-1B, 1E), other proteins elicit UPR already at low dosage (Figure 1C-1E). Thus, the threshold for activation of transcriptional programs in response to increasing burden of misfolded protein (Figure 2) must depend on intrinsic features of the accumulating polypeptide. We will couple genome wide gene expression profiling (in collaboration with F. Bertoni, IOR, Bellinzona) with proteome analysis, shotgun and selected reaction monitoring-based (SRM) proteomics (in collaboration with P. Picotti, ETH Zurich) in order to establish transcriptional and post-translational cellular response “fingerprints” associated to individual defective polypeptide expression and drug treatments. Responses to accumulation of misfolded proteins below the threshold required for UPR activation will be analyzed with particular care. These responses that we collectively termed ERAD tuning could rely on post-translational mechanisms, which have much shorter latency, since they do not depend on gene transcription and translation (e.g. modulation of ER-resident proteins turnover, formation/disassembly of functional complexes, sub-compartmental de-localization, post-translational modifications such as ADP-ribosylation, palmitoylation, …). Some of these non-transcriptional responses that regulate ER-resident proteins level and activity are hijacked by human pathogens during their infection cycle.

Figure 1: Substrate-dependent ER stress-induction. (A) Tet-induced expression of CD3δ-HA. BiP, GRP94 and HERP levels. Last lane, Tun-induced ER stress (5µg/ml). (B) Variations of CD3δ (fmol protein/ cell) and BiP transcripts. (C)-(D) same as (A)-(B) for BACE457. (E) Induction of stress markers at maximal CD3δ and BACE457 doses and at 5µg/ml Tun, quantitative real time PCR.

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2: Induction of non-transcriptional and transcriptional UPR. Threshold (dotted line) shows the level of model protein that triggers a transcriptional UPR (UPRTr). Below the threshold, responses, if any, are not-transcriptional, and ill-defined.

Noack et al., J Virol. 2014; 88:10272-10275.

Noack et al., Nat Chem Biol. 2014; 10:881-882.

Noack et al.,Swiss Med Wkly. 2014; 144:w14001.