Proinsulin folding and degradation in the Marzec Group
Our focus in the Marzec Group is to understand the mechanisms necessary for efficient proinsulin folding, the role of endoplasmic reticulum chaperones in the process and the relationship to the diabetes pathology.
Our mission is to uncover basic biological processes related to protein folding and harness them to formulate new therapeutic approaches.
We value working environment that combines an ambitious and a social atmosphere with high academic standards, open collaborations and innovation.
Our research is focused on uncovering discrete but critical steps in proinsulin folding in the endoplasmic reticulum (ER). Multiple studies during the last decades established major transition points during the processing and formation of mature insulin e.g. removal of preproinsulin’s signal peptide, intramolecular disulphide bonds formation and the removal of proinsulin’s C-peptide by proteases.
Yet, many fundamental questions remain to be answered.
Pancreatic β cells are highly specialized secretory cells that respond to fluctuating insulin demands. It is highly probable that they have evolved dedicated adaptive mechanisms that enable on one hand increased synthesis, processing, quality control and secretion of insulin and on the other hand quick degradation via multiple outputs.
What are those mechanisms? Does proinsulin folding require more than just disulphide bond formation? What proteins take part in this process? Is folding quality control restricted to endoplasmic reticulum?
Certain mutated and differentially folded proinsulins escape the quality control of ER. They present normal disulphide bond formation but are characterized by discrete structural, folding and bioactivity differences. How do they escape the ER quality control? Do such misfoldings contribute to β cell demise during increased insulin production or do additional quality control mechanisms exists and is β cells protected by it?
We are the first group that described proinsulin chaperone, termed GRP94 (or Endoplasmin, gp96), required for proper proinsulin processing (Ghiasi et al.). This work follows the identification of GRP94 as a critical chaperone, evolutionary and structurally linked to proinsulin, Insulin-like Growth Factor 1/2 (Wanderling et al.), suggesting GRP94 as the chaperone of a family of insulin-like peptides. However, the involvement of other endoplasmic reticulum chaperones, except of disulphide isomerases, in proinsulin folding remains under investigated and is currently one of our main focuses.
Multiple studies have established Endoplasmic-reticulum-associated protein degradation (ERAD) as the main degradation pathway for misfolded proinsulin. It has long been assumed that proinsulin final degradation is performed by standard proteasome. However recently, we have shown that non-standard proteasome subunits are expressed by β cells, form active proteasomes and participate in proinsulin degradation. Additionally, their expression is increased when folding capacity of β cells is diminished or by low doses of cytokine IL1β (Khilji et al. and 2nd manuscript in submission). As non-standard proteasomes differentially process polypeptides for MHC presentation, changes in proteasome composition in response to external stimuli could induce β cell directed autoimmune response, a feature of Type 1 diabetes.
Finally, a bulk of insights on proinsulin folding has been gathered via the employment of proinsulin mutants e.g. AKITA. As valuable as they are, it is important to note that the vast majority of diabetes patients do not carry mutations in the insulin gene. It is therefore reasonable to ask if terminal misfolding of a mutated proinsulin and its subsequent degradation, report on mechanisms relevant for wild-type proinsulin and thus majority of patients? And if not, will development of models where deficient folding environment induces misfolding of wild-type proinsulin be more appropriate to investigate the physiologically-relevant aspects of β cells behaviour? So far, such an approach has been hampered by the lack of folding deficient models but with the advent of CRISPR/Cas technology we are now well equipped to create and investigate them. An example of such an approach is our knock-out of GRP94 chaperone in INS1-E β cells that results in almost complete proinsulin loss and its degradation by non-standard proteasome.
In the end, we believe that future therapies may not only correct β cell physiology to sustain insulin production but utilize folding mechanisms to produce in vivo insulin(s) with desired biological activities e.g. fast- or slow-acting molecules. As such, our research intends to further the underlying mechanisms of proteins misfolding and handling of misfolded proteins specifically in a β cell setting. We believe that this research can be used to improve efficiency and quality of current diabetes treatments as well as developing new treatments. Translationally, we hope that any and all discovered mechanisms of protein mishandling could be implemented in other protein misfolding diseases, and as such help not just diabetic patients, but also e.g. Alzheimer’s patients.
The key achievements since the establishment of our group at BMI in 2015 include:
- Description of the first proinsulin chaperone, namely GRP94.
- Discovery that non-standard proteasomes are expressed in β cells and participate in proinsulin degradation.
- Finding that β cells with diminished folding capacity overexpress non-standard proteasomes in order to efficiently degrade increased amount of misfolded proinsulin.
Postdocs and PhD students are welcomed to contact us about the potential collaboration, joint grant application or internship. We are always looking for highly motivated Master's and ERASMUS students to work with us.
All incoming lab members will participate in project design and execution, fundraising (grant writing), writing scientific reviews and, if possible, attend scientific meetings and present posters.
We foster independence from the start and provide the opportunity to grow and excel in majority of research relevant subjects.
- Description of novel proinsulin folding steps and chaperones involved in the process.
- The role of non-standard proteasomes in proinsulin degradation.
- Hyperinsulinemia: protein chaperones as novel therapeutic targets.
The following techniques are in use in the lab:
- Protein analysis (reducing and non-reducing SDS-PAGE, Western blotting, immunoprecipitation, mass spectrometry, size-exclusion chromatography)
- Plasmid design to express protein mutants, cloning, bacterial transformation, cell transfection,
- lentivirus production and cell transduction
- Gene knock-down or -out (siRNA, shRNA, CRISPR-Cas9),
- gene expression (RT-qPCR)
- Advanced imaging that includes confocal microscopy, electron microscopy at local CFIM,
- viability and functional assays e.g. proteasome activity,
- Cell- and islet-culture, islet isolation, pancreatic islet handling and usage
and many more, depending on the needs of the project.
Prof. Thomas Mandrup-Poulsen, UCPH, Denmark, scope: metabolic and inflammatory conditions influencing proinsulin folding
Prof. Peter Arvan U. of Michigan, USA, scope: proinsulin misfolding and disulphide bond formation
Prof. Pontus Gourdon, Per Amstrup Pedersen and Kamil Gotfryd, UCPH, Denmark, scope: structural aspects of proinsulin folding complex
Prof. Anders Tengholm, Uppsala University, Sweden, scope: pro- and insulin activity in β cells with diminished folding capacity
Prof. Micheal Davies and Per Hagglund, UCPH, Denmark, scope: mass spectrometry based protein identification in various β cell samples
Prof. Flemming Pociot Herlev-Gentofte Hospital, Denmark, scope: genetic variations in type 1 diabetes
Prof. Ewa Gurgul-Convey Hannover Medical School, Hannover, Germany, scope: MCPIP21 functions in β cell biology
- EFSD/Lilly Programme
- Magda Sofie og Aase Lütz Mindelegat
- Private Danish Foundations for purchase of lab equipment (OMIX application)
- DFF FTP Project 1 (Prof. Gourdon PI)
- Augustinus Fonden
- Vissing Fonden
- Bjarne Jensen Fonden
- Poul og Erna Sehested Hansens Fond
- EFSD/Lilly Programme
- Eva og Hans Carl Holms Mindelegat
- Kirsten og Freddy Johansens Fond
- A.P. Møller Fond
- Dagmar Marshall Fond
- Augustinus Fond
- DFF Marie Curie Mobilex
- K01 NIH, National Institute on Aging
Muhammad Saad is employed as PhD student at the Department of Biomedical Sciences, University of Copenhagen. Earlier he earned his bachelors (Doctor of Veterinary Medicine) and Masters (Physiology) from Pakistan.
The focus of Saad’s research is on auto-immune mechanisms related to type-1 diabetes. He will explore the role of GRP94 in processing of pro-insulin. In addition, he will investigate the potential involvement of proteasomes in proinsulin degradation.
Currently Saad is on an exchange visit to Anthony Purcell laboratory at Monash University, Australia.
Celina Phil (Bsc in Molecular Biomedicine) is currently doing her MSc in Molecular Biomedicine. Celina wrote her bachelor’s thesis with Associate Professor Marzec, writing about GRP94, ER stress and degradative pathways.
As a continuation of work previously done in the lab, Celina is now focusing her research on proteins co-interacting with GRP94 and proinsulin. The goal is to give a comprehensive picture of the relationship between ER chaperones and modulating proteins, and to investigate their collaborative effect on insulin biosynthesis and processing.
Sophie Emilie Bresson (Bsc. In Molecular Biomedicine, UCPH) is working with Associate Professor Marzec, in order to complete her Master’s degree in Molecular Biomedicine.
Sophie has been working with the group since January 2018, where she as a bachelor student investigated the interaction between endogenous GRP94 and proinsulin, with focus on identifying novel interaction partners.
Today, Sophie’s work centers around GRP94 and proinsulin variants, investigating their effect on complex formation, protein processing and maturation, ER-stress, and its relation to diabetes.
Tenna Holgersen Bryde (M.Sc. in Cell Biology and Physiology, UCPH) is employed as a research assistant at the Department of Biomedical Sciences, University of Copenhagen.
Tenna has been working with Associate Professor Marzec since she joined his group in the fall of 2018, where she finished her Master’s studies exploring proteins co-interacting with GRP94 and proinsulin.
The main objective of Tenna’s work as a research assistant is a continuation of her studies from M.Sc. She will further the research to understand the mechanisms necessary for efficient proinsulin folding and their relationship to diabetes pathology.