Periodic Reporting for period 1 - CytoLysoReg (Cellular and Biophysical Approach to Understand the Cytokine-mediated Regulation of the Lysosomal Hydrolytic Capacity)
Reporting period: 2021-09-01 to 2023-08-31
Dysfunction of lysosomes partakes the onset of multiple human pathologies. Even though the activity of lysosomal hydrolases was considered confined to the lysosomal lumen for more than 50 years, nowadays we know they localise in the nuclear and cytosolic compartment. These extra-lysosomal activities have exponentially increased the number of biological processes controlled by lysosomes and their potential contribution to the onset and progression of human diseases. Thus, a deeper understanding of the signalling pathways involved in the regulation of lysosomes would provide new tools to ameliorate the symptoms associated with these diseases.
Lysosomes are the recycling plants of cells, but additionally take part in diverse features of cell life. Therefore, there is a strong interest in understanding the mechanisms that control its activity. The discovery of the role played by the mTOR-TFEB axis during starvation served to unveil partially the regulation of lysosomal gene expression. However, as recently shown by the researcher and others, the cytokines-STAT3 axis is also important to lysosomal governance. However, the molecular mechanisms of the cytokine-mediated lysosomal regulation remain unresolved.
CytoLysoReg implementation has served to unveil kinetic differences between the IL-6 and IL-10 signalling networks explaining the ability of IL-6 to induce the expression of lysosomal proteases, contrary to IL-10. First difference is the transient activation of STAT3 and STAT1 by IL-6 vs. persistent STAT3 activation and lack of STAT1 activation by IL-10. The second, a sustained activation of STAT3 obtained with IL-6 not elicited by IL-10. However, it falls short to explain the molecular mechanism through which IL-10 inhibits their expression. Then, our phospho-proteomics approach has allowed us to dig deeper in the differences between these two cytokines. Meanwhile, IL-6 induces the phosphorylation and activation of signalling pathways and proteins regulating transcription, gene expression and proliferation, therefore further enhancing the expression of these lysosomal proteases; IL-10 inhibits these pathways thus explaining the inhibitory effect of IL-10 on lysosomal activity. This information provides us with novel targets involved in the cytokine-mediated regulation of the lysosomal compartment, thus offering new tools to fine-tune lysosomal activity.
On the other hand, Oncostatin M—a member of the IL-6 cytokine-family—has been shown to regulate LM-PCD through STAT3. Notably, recent data link STAT3 to the upkeep of lysosomal homeostasis without LM-PCD induction. However, the molecular mechanisms explaining this dichotomic behaviour of STAT3 have not been elucidated. Through CytoLysoReg implementation we are currently generating a phospho-proteomic dataset comparing the signalling pathways and biological processes regulated during LM-PCD and lysosomal homeostatic response that will allow us to unveil how STAT3 can switch between a pro-survival factor controlling lysosomal homeostasis and a trigger of LM-PCD
Lysosomes are the recycling plants of cells, but additionally take part in diverse features of cell life. Therefore, there is a strong interest in understanding the mechanisms that control its activity. The discovery of the role played by the mTOR-TFEB axis during starvation served to unveil partially the regulation of lysosomal gene expression. However, as recently shown by the researcher and others, the cytokines-STAT3 axis is also important to lysosomal governance. However, the molecular mechanisms of the cytokine-mediated lysosomal regulation remain unresolved.
CytoLysoReg implementation has served to unveil kinetic differences between the IL-6 and IL-10 signalling networks explaining the ability of IL-6 to induce the expression of lysosomal proteases, contrary to IL-10. First difference is the transient activation of STAT3 and STAT1 by IL-6 vs. persistent STAT3 activation and lack of STAT1 activation by IL-10. The second, a sustained activation of STAT3 obtained with IL-6 not elicited by IL-10. However, it falls short to explain the molecular mechanism through which IL-10 inhibits their expression. Then, our phospho-proteomics approach has allowed us to dig deeper in the differences between these two cytokines. Meanwhile, IL-6 induces the phosphorylation and activation of signalling pathways and proteins regulating transcription, gene expression and proliferation, therefore further enhancing the expression of these lysosomal proteases; IL-10 inhibits these pathways thus explaining the inhibitory effect of IL-10 on lysosomal activity. This information provides us with novel targets involved in the cytokine-mediated regulation of the lysosomal compartment, thus offering new tools to fine-tune lysosomal activity.
On the other hand, Oncostatin M—a member of the IL-6 cytokine-family—has been shown to regulate LM-PCD through STAT3. Notably, recent data link STAT3 to the upkeep of lysosomal homeostasis without LM-PCD induction. However, the molecular mechanisms explaining this dichotomic behaviour of STAT3 have not been elucidated. Through CytoLysoReg implementation we are currently generating a phospho-proteomic dataset comparing the signalling pathways and biological processes regulated during LM-PCD and lysosomal homeostatic response that will allow us to unveil how STAT3 can switch between a pro-survival factor controlling lysosomal homeostasis and a trigger of LM-PCD
Objective 1. Cytokine-mediated Regulation of Lysosomal Activity
Preliminary data previously generated by the researcher during his work as Senior Research Associate at the University of Dundee (UK) showed IL-6 stimulation induces changes in the levels of proteins in the endolysosomal compartment, regulating multiple signalling pathways, suggesting cytokines regulate multiple signalling pathways that could be involved in the regulation of the lysosomal compartment. To understand the molecular mechanisms behind the opposite role that pro- (i.e. IL-6) and anti-inflammatory (i.e. Interleukin-10 (IL-10)) exert on the lysosomal compartment a comprehensive approach has been followed. The role of these two cytokines on the lysosomal compartment of human T cells (i.e. Jurkat T cells) has been analysed by following over the time the activation of different signalling pathways upon cytokine stimulation, the activity of different lysosomal proteases by in vitro (i.e. using specific substrates) and in cell (i.e. using DQ-BSA as a way of measuring changes in the proteolytic capacity of the lysosomal compartment) activity assays and immunofluorescence, and changes in their levels of expression by immunoblotting and qPCR analysis. This approach has allowed us to get a detailed picture of how pro- and anti-inflammatory cytokines affect the lysosomal compartment. Finally, to understand the different signalling properties allowing these two groups of cytokines to play different roles in lysosomal biology we resorted to phospho-proteomics analysis upon stimulation with IL-6 or IL-10 for 15 min and 9 h, to distinguish between early and late events triggered by cytokine stimulation.
Objective 2. Developing New Tools Against Breast Cancer
LM-PCD elicitor-activity of Oncostatin M opens new avenues for breast cancer therapy. However, it is known that pH values —acidic in the cancer environment—affect signalling. Therefore, engineering OSM variants with higher binding affinity towards its cognate receptor may allow us to use them as therapeutical targets to treat breast cancer. For this purpose, the signalling properties of OSM in breast cancer cell models (i.e. MDA-MB-231 and SkBr3) has been studied. Furthermore, the changes induced by OSM in the levels of expression of selected lysosomal proteases, both at the protein and the mRNA level, have been analysed. This has allowed us to distinguish between changes at the proteomic level (e.g. changes in protein stability) and changes occurring at the transcriptomic level because of cytokine stimulation. Finally, an error-prone library of OSM variants has been built and screened for variants with higher binding affinities towards the receptors.
During the implementation of this Action our work was presented at an international meeting (Plenary talk, FEBS/IUMBM CrossMitoNus Meeting, March 2022, Seville) at the 20th Science Fair (May 2022, Seville) and has contributed to the publication of three manuscripts.
Preliminary data previously generated by the researcher during his work as Senior Research Associate at the University of Dundee (UK) showed IL-6 stimulation induces changes in the levels of proteins in the endolysosomal compartment, regulating multiple signalling pathways, suggesting cytokines regulate multiple signalling pathways that could be involved in the regulation of the lysosomal compartment. To understand the molecular mechanisms behind the opposite role that pro- (i.e. IL-6) and anti-inflammatory (i.e. Interleukin-10 (IL-10)) exert on the lysosomal compartment a comprehensive approach has been followed. The role of these two cytokines on the lysosomal compartment of human T cells (i.e. Jurkat T cells) has been analysed by following over the time the activation of different signalling pathways upon cytokine stimulation, the activity of different lysosomal proteases by in vitro (i.e. using specific substrates) and in cell (i.e. using DQ-BSA as a way of measuring changes in the proteolytic capacity of the lysosomal compartment) activity assays and immunofluorescence, and changes in their levels of expression by immunoblotting and qPCR analysis. This approach has allowed us to get a detailed picture of how pro- and anti-inflammatory cytokines affect the lysosomal compartment. Finally, to understand the different signalling properties allowing these two groups of cytokines to play different roles in lysosomal biology we resorted to phospho-proteomics analysis upon stimulation with IL-6 or IL-10 for 15 min and 9 h, to distinguish between early and late events triggered by cytokine stimulation.
Objective 2. Developing New Tools Against Breast Cancer
LM-PCD elicitor-activity of Oncostatin M opens new avenues for breast cancer therapy. However, it is known that pH values —acidic in the cancer environment—affect signalling. Therefore, engineering OSM variants with higher binding affinity towards its cognate receptor may allow us to use them as therapeutical targets to treat breast cancer. For this purpose, the signalling properties of OSM in breast cancer cell models (i.e. MDA-MB-231 and SkBr3) has been studied. Furthermore, the changes induced by OSM in the levels of expression of selected lysosomal proteases, both at the protein and the mRNA level, have been analysed. This has allowed us to distinguish between changes at the proteomic level (e.g. changes in protein stability) and changes occurring at the transcriptomic level because of cytokine stimulation. Finally, an error-prone library of OSM variants has been built and screened for variants with higher binding affinities towards the receptors.
During the implementation of this Action our work was presented at an international meeting (Plenary talk, FEBS/IUMBM CrossMitoNus Meeting, March 2022, Seville) at the 20th Science Fair (May 2022, Seville) and has contributed to the publication of three manuscripts.
Lysosomes are nowadays recognised players in the regulation of a plethora of biological processes through both their intra- and extra-lysosomal activities, therefore involved in the onset and progression of multiple human pathologies. However, our narrow understanding of the signalling pathways controlling lysosomes is currently limiting our capacity to fully exploit their potential as therapeutical targets. STAT3 has been shown to be able to induce the expression of lysosomal genes. However, an opposite role for pro- and anti-inflammatory cytokines has been shown regarding lysosomal regulation, despite both cytokines inducing STAT3 activation. Our multiple approach—by combining molecular, cellular, genomics and proteomics—has allowed us to dissect the dichotomic effect both cytokines play in the regulation of the lysosomal compartment. Thus, differential signalling kinetics combined with the opposite regulation of transcription, gene expression and proliferation accounts for this dichotomic effect. On the other hand, the signalling properties of OSM—a member of the Il-6 family of cytokines—known to act as a trigger of LM-PCD have been evaluated in comparison to stimulus triggering lysosomal homeostatic responses by combining cellular and proteomic studies. This data will allow us to unveil the molecular mechanisms tilting STAT3 from its role as a keeper of lysosomal homeostasis into an inducer of LM-PCD.