The Protein Chemistry Laboratory (PCL) was established in 2009 at the college of sciences of Shiraz University. More than a decade ago, the activity of this laboratory started by studying the structural and functional analyses of eye lens crystallins due to genetic mutations or chemical changes after post translational modifications. Crystallin proteins with a dominant β-sheet secondary structure and high stability have extremely high concentrations in the lenticular tissue, and some of them, such as αB-crystallin, have a wide tissue distribution. These proteins are divided into α-crystallin (oligomer with structural function, chaperone and anti-apoptotic activities), β-crystallin (oligomer with mainly structural function) and γ-crystallin (monomer with structural function). The changes in their structures and even a minor alteration in the subtle interaction among different crystallin proteins may strongly affect the quality of vision, so that these proteins are important pathogenic targets for mutations and damaging reactions with highly reactive and destructive molecules which their concentration may rise in the body during some diseases, especially  in Diabetes Mellitus (DM).

Numerous genetic mutations have also been reported in the genes of two subunits of human α-crystallin (αA- and αB subunits) that are associated with cataracts and myopathy, or both. Therefore, one of the important research interests in our laboratory is to create these mutations by site directed mutagenesis in genes of human recombinant crystallin proteins and to discover and explain the biochemical mechanism of their pathogenesis in various ways and by the aid of different tools.

Optimizing the laboratory production of pharmaceutical proteins and peptides such as human recombinant insulin, proinsulin and anti-diabetic incretin peptides and creating targeted mutations in their genes with the aim of introducing new drug analogues, possessing improved bioactivity and stability is another major goal in the PCL. One of the important strategies for industrial production of human insulin is through the bacterial expression and production of the recombinant proinsulin, which is very similar to its production in the beta cells of the Langerhans islets. Also, one of the main challenges of pharmaceutical insulin, which is first produced from the pancreas of animals such as cattle and pigs and then by recombinant DNA technology based on nucleotide sequence of human insulin gene, is its high instability and susceptibility for fibrillation/aggregation under physical and chemical stresses. Today, with a slight change in the amino acid sequence of human insulin, new types of insulin called insulin analogues such as Glargine (Lantus), Lispro, Aspart, NPH, Detemir, and Glulisine have been produced, which are rapidly replacing the regular insulin in the pharmaceutical market.

Therefore, another major interest in the PCL is the laboratory production of new types of insulin analogues and the study of their structure, stability, and bioactivity.

More than 50% of the insulin secreted by the pancreas is due to the action of incretin peptides such as GLP1 and GIP, which are produced from the intestinal endocrine cells. Due to the high instability and low half-life of the endogenous incretins, an important alteration has been made in the amino acid sequence of their pharmaceutical forms. Another research interest in our laboratory is the optimization of laboratory production and the study of the structure and function of new incretin anti-diabetic medicines.

α-glucosidase enzyme is an important target for inhibition by anti-diabetic medicines such as Acarbose, but inhibition of the enzyme by this anti-diabetic drug is associated with serious and important complications and side effects. Therefore, the introduction of new chemical compounds that inhibit this enzyme more effectively and have fewer side effects is of great medical importance.

 Cisplatin is an organometallic complex that has been used to treat various types of cancer, but its medical application is associated with serious complications and problems in the patients. Therefore, the design of new platinum derivatives that have improved activity and less harmful side effects are welcomed by the medical community. In the past decade, several research projects have also been carried out in our laboratory in collaboration with Department of Chemistry of Shiraz University, the result of which has been the introduction of several new inhibitory compounds against α-glucosidase enzyme or a number of novel platinum complexes with an appropriate and improved anti-cancer activity.

 

Methods and tools

  1.  Primer designing, site directed mutagenesis and gene cloning.
  2.  Confirming performed mutations by DNA nucleotide sequencing.
  3.  Agarose gel electrophoresis of PCR products.
  4.  Optimizing expression of recombinant proteins and peptides in prokaryotic host system
  5.  Purification of recombinant proteins and peptides using a variety of chromatographic methods. 
  6.  Purity analysis of proteins and peptides by HPLC and SDS-PAGE.
  7.  Identification of proteins and peptides by mass spectrometry and Western blot analyses.
  8.  Studying the secondary and tertiary structures of proteins and peptides by fluorescence, circular dichroism (CD), FTIR and Raman spectroscopies.
  9.  Studying the quaternary structure (oligomerization state) of mutant α-crystallins using dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and analytical ultra-centrifugation (AUC)
  10.  Assessment of thermal stability of proteins by DSC⸲ fluorescence and CD methods.
  11.  Analysis of chemical stability of proteins by fluorescence method.
  12.  Evaluation of proteolytic stability of proteins and peptides by gel electrophoresis.
  13.  Studying chaperone activities (in vitro and in vivo) of the mutant α-crystallins by UV-Vis spectroscopic method and colonies formed by the bacterial host cells.
  14.  Evaluation of α-glucosidase enzymatic activity as target for the chaperone activity of the mutant α-crystallins  and potential antidiabetic compounds by UV-Vis spectroscopic method.
  15.  Analysis of refolding ability of mutant α-crystallins by UV-Vis spectroscopic method.
  16.  Studying fibrillation and amorphous aggregation of mutants crystallins and mutant insulins by spectroscopic and microscopic approaches (transmission electron microscope (TEM) and fluorescence microscope).
  17. Toxicity study of the amyloid fibrils formed by mutant forms of human recombinant insulin and α-crystallin using MTT assay. Apoptosis inducing activity assessments of organometallic complexes and other synthetic or natural compounds. 
  18. Animal studies: studying the biological activities of insulin and incretin peptides.