Folding

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.

Degradation

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 2^nd 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.

Investigative models

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:

1. Description of the first proinsulin chaperone, namely GRP94.
   
   
2. Discovery that non-standard proteasomes are expressed in β cells and participate in proinsulin degradation.
   
   
3. Finding that β cells with diminished folding capacity overexpress non-standard proteasomes in order to efficiently degrade increased amount of misfolded proinsulin.

1. Description of novel proinsulin folding steps and chaperones involved in the process.
2. The role of non-standard proteasomes in proinsulin degradation.
3. 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 Godfryd, 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

Our funding

Present:

• 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)

Past:

• 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