Role of Conformational Changes in human P450 Oxidoreductase (POR) on the metabolic regulation.
Mutations in human NADPH cytochrome P450 oxidoreductase (POR) cause metabolic disorders. However, we have a very limited understanding of how POR mutations result in aberrant metabolism. The mechanistic details of the interplay between biochemical inputs, allosterically coupled protein domains, and downstream effectors underlying POR activity and plasticity, which are crucial for pharmacology and metabolic control, remain poorly understood. In this project we aim to explore the role of structural dynamics on POR activity and plasticity and dissect how pathogenic mutations and regulatory inputs control POR functional phenotype; and, capitalize on these insights to explore novel ligands that target POR, to control the metabolism with the ultimate aim of reversing metabolic disorders.
Unveiling the existence of time-dependent activity fluctuation and distribution of activities between seemingly identical proteins is a phenotype that remains masked in conventional averaging kinetics. The potential of direct and synchronous observation of structural and functional dynamics of an enzyme at work promises to uncover how structural dynamics encode biological function – the fundamental language of protein biology. By combining genetic, cellular, enzymatic, and biophysical methods, our studies will provide a molecular understanding of the role of protein conformational dynamics in redox biology and electron transfer patterns that govern drug and xenobiotic metabolism in humans. Collaborations with Denmark and Portugal are in place for this project,
Regulation of Androgen Production in Humans by Control of CYP17A1 Activities
Androgen production is a target for the treatment of several metabolic disorders. Human androgens are made from dehydroepiandrosterone (DHEA). CYP17A1 catalyzes DHEA production by its 17,20 lyase activity. The 17,20 lyase activity of CYP17A1 requires cytochrome b5 (CYB5A). The lyase activities of CYP17A1 are regulated by CYB5A; and protein phosphorylation, therefore, the tissue-specific regulation of these factors may provide a mechanism for the regulation of CYP17A1 lyase activity. These studies also provide details about regulation of human sex steroid production and help in understanding hyperandrogenic disorders and identify novel mechanisms to target in prostate cancer.
Human Genetic Variations in P450 oxidoreductase POR
A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). In 2004 the first human patients with defects in POR were reported by us and others, and currently over 300 variations in POR are known. As the energy supplier to majority of cytochrome P450 enzymes, effects of mutations in POR reach far beyond steroid biosynthesis and POR variants manifest with a very broad phenotype, like a chameleon changing color depending on its surrounding. We are creating personalized models of POR mutations found in patients as well as in normal population using CRISPR/CAS and modified bacterial cells to study steroid and drug metabolism.
These studies will establish the functional assessment of POR mutations in metabolic processes and help with diagnosis and treatment by supplements. A large number of collaborations from multiple countries including France, Spain, Argentina, UK, USA, Canada and elsewhere are in place, since our laboratory is a central reference laboratory for research on POR genetic variants.
Novel Chemicals for the Treatment of Prostate Cancer and Polycystic Ovary Syndrome.
Overproduction of androgens is linked to the pathogenesis of prostate cancer and polycystic ovary syndrome. Steroidogenesis, and more specifically, the 17,20-lyase reaction catalyzed by cytochrome P450 17α-hydroxylase/17,20- lyase and Aldo-keto reductase AKR1C3 have become topics of interest due to the clinical importance of these key steroidogenic enzymes. The current anti-prostate cancer drug of choice, abiraterone that targets CYP17A1, is not very effective and has severe side effects. We have previously shown that Abiraterone also inhibits CYP21A2, thus inhibiting cortisol production, and a metabolite of abiraterone is a strong androgen which ultimately defeats the treatment. A better CYP17A1 inhibitor (preferably non-steroidal) is needed. In this collaboration project with computational and medicinal chemistry groups from Denmark, Poland, Italy, Spain and Austria to produce novel inhibitors of CYP17A1 and AKR1C3 we design and improve the compounds andl test them in the laboratory. After the virtual screening, we apply machine learning and automated workflows to identify pharmacophores for structural modifications and synthesis of novel chemicals. Using several cell and recombinant protein models we test the efficicasy of novel inhibitors.
Targeting ACTH Receptor for the Treatment of Congenital Adrenal Hyperplasia
Congenital adrenal hyperplasia (CAH) is the common disorder of steroid production mainly caused by a mutated CYP21A2 gene, causing 21-hydroxylase deficiency (21OHD). CAH due to 21OHD requires lifelong mineralocorticoid (MC) and glucocorticoid (GC) therapy. But, from location of the enzyme block in the steroid production and increase of adrenocorticotropic hormone (ACTH) which stimulates the melanocortin type 2 receptor (MC2R) in the adrenal, excessive adrenal androgen production remains a challenging side effect for CAH even under MC and GC treatment. In CAH, ACTH regulation of glucocorticoids through MC2R receptor causes androgen excess. Side effects of this can be reduced by inactivating MC2R receptor in patients with CAH. In this project we design, test and optimize peptides that act as potent MC2R antagonists/agonists. Overcoming the androgen overproduction in CAH is a fundamental problem in medicine. Currently, there is no treatment available to block MC2R and solve the problem of androgen excess in CAH treatment.
Disorders of Human Growth and Genetic Variations in Human Growth Hormone Gene GH1
Isolated Growth Hormone (GH) deficiency (IGHD), is the common form of growth disorder related to pituitary hormone defects and results in growth failure in children. In isolated GHD (IGHD), mutations in several known genes like growth hormone (GH-1), growth hormone receptor (GHR), growth hormone releasing hormone receptor (GHRHR) or transcription factors involved in pituitary development, have been identified in relatively small percentage of patients suggesting that many other genetic and environmental causes may affect growth. In IGHD II, the autososomal dominant form of GHD, mutations occurring in and around exon 3, lead to aberrant splicing. These mutations include splice site mutations as well as disruption of splicing enhancers that are necessary to promote GH-1 constitutive splicing. The common feature of all these GH-1 gene alterations is the loss of exon 3 from the mRNA during splicing producing the 17.5-kd isoform. The 17.5-kd isoform is retained in the endoplasmic reticulum, disrupts the Golgi apparatus, and impairs secretory protein trafficking exerting a dominant-negative effect on the secretion and stability of the 22-kd isoform in both tissue culture and in transgenic animals. Despite some common features, clinical phenotype can vary widely depending on the individual genotypes and reflects a threshold and a dose dependent effect of the amount of the 17.5-kd relative to 22-/20-kd transcript ratio. In this project we are exploring the complexities of GH-1 genetics and pathophysiology, and find novel mechanisms for GH regulation, transport and secretion that ultimately regulate human growth. The focus of our studies is to find approaches to rescue IGHD II. Collaborations with Brazil, UK and USA are in place for this project.
Analysis of Human Genetic Variations in CYP21A2 for Links to Congenital Adrenal Hyperplasia
Most patients with Congenital adrenal hyperplasia (CAH) suffer from the deficiency of steroid 21-hydroxylase (21OH) due to mutations in the CYP21A2 gene. More than 300 mutations have been found in the CYP21A2 alleles. The detection and characterization of theses mutation has continued to expand worldwide, since these studies improve the correct CAH diagnosis, appropriate treatment and genetic counseling. Therefore, the aim of this project is to investigate functional consequences of CYP21A2 gene mutations using in vitro assays and to correlate the functional consequences to CAH phenotypes. Mutated CYP21A2 expression constructs are transiently expressed in cells and enzyme activities are determined by metabolism of the 21OH substrates (17-hydroxyprogesterone or progesterone) to the corresponding products (11-deoxycortisol or deoxycorticosterone). These studies provide molecular basis of disease for the patients with CYP21A2 mutations and improve our current understanding of 21-hydroxylase deficiency and CAH. Collaborations with Brazil and France are in place for this work.
