Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Insulin production and maturation are typically confined to specialized pancreatic beta cells.
• Proinsulin requires specific enzymes for conversion into mature insulin.
• The constitutive secretory pathway is present in most cell types.

Purpose of the Study:

• To engineer human proinsulin for processing in non-specialized cells.
• To enable insulin production via the constitutive secretory pathway.
• To investigate mutations enhancing insulin accumulation and bioactivity.

Main Methods:

• Site-directed mutagenesis to introduce furin consensus cleavage sequences into human proinsulin cDNA.
• Utilizing the constitutive secretory pathway for proteolytic maturation.
• Measuring mature insulin accumulation via radioimmunoassay.
• Assessing insulin bioactivity through insulin receptor autophosphorylation assays.

Main Results:

• Engineered proinsulin with furin cleavage sites underwent efficient maturation in cells with only a constitutive pathway.
• A specific mutation (HisB10 to Asp) significantly increased mature insulin accumulation (10- to over 100-fold).
• Secreted peptides matched the molecular weights of insulin A and B chains, indicating correct processing.
• The produced insulin demonstrated bioactivity by stimulating insulin receptor autophosphorylation.

Conclusions:

• Human proinsulin can be engineered for efficient processing and maturation in the constitutive secretory pathway.
• Specific mutations can dramatically enhance the accumulation of mature, active insulin.
• This approach allows for the potential engineering of any cell type to produce stable, active insulin.