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White adipose tissue (WAT) is the primary site in the body for storing triglycerides. WAT distribution and function vary based on sex, and accordingly, disorders in which WAT plays a role, including obesity, type two diabetes, and metabolic syndrome, have sex differences in risk factors and etiologies. Prohibitin 1 (PHB1) is a protein involved in adipocyte-intrinsic activities sensitive to sex hormone signaling. PHB1 has a bidirectional relationship with estrogens and androgens, partly mediated by tyrosine phosphorylation. Another post-translational modification site in PHB1 is Cys69. It is the only cysteine residue in the amino acid sequence of the Phb1 gene, and it is essential in targeting PHB1 to plasma membranes in adipocytes. To investigate the role of Cys69 at a systemic level, we developed a knock-in mouse model replacing Cys69 with an alanine in Phb1. These are known as the Phb1-KiC69A (or Phb1-Ki) mice. Based on what is known about PHB1, I hypothesized that these mice would display sex differences in their WAT phenotype. This study's objectives were to define changes in the WAT phenotype in the male and female Phb1-Ki mice compared to wild-type mice, and to determine whether any differences displayed sex-specificity. Visceral (VAT) and subcutaneous (SAT) adipose tissue was collected from wild-type and Phb1-Ki male and female mice. They were compared based on histology, gene and protein expression, and PHB1 subcellular localization. Primary cells from VAT and SAT were also collected and differentiated into adipocytes, and lipolysis and lipid uptake were measured. The serum concentrations of three adipokines involved in metabolic regulation were analyzed. The results showed that Phb1-Ki mice have sex- and depot-specific changes compared to wild-type mice in lipid droplet size, tissue mass, PHB1 subcellular localization, and adipocyte protein expression levels. Resistin levels are also decreased in both sexes of Phb1-Ki mice compared to wild-type. Adipocytes

Prohibitin-1 (PHB1) is a highly conserved protein that plays a crucial role in regulating cell proliferation, apoptosis, mitochondrial stability, and cell signaling processes. PHB1 undergoes various post-translational modifications (PTMs), enabling it to execute cell compartment-specific and pleiotropic functions. One such PTM site is cysteine 69 (Cys69) residue, which is conserved among vertebrates. Our laboratory developed a knock-in mouse model (Phb1-Ki), substituting Cys69 with an alanine residue, to explore the role of Cys69 in PHB1’s pleiotropic functions, including immune function. The Phb1-Ki mice displayed changes in spleen and thymus weights, and their histological analysis showed morphological differences when compared with age and sex matched wild-type control mice. The flow cytometry experiments revealed a decrease in CD4+ and CD8+ cell populations in the spleen of the male and in the thymus of female Phb1-Ki mice compared with respect to wild-type mice. To further understand PHB1's role in T cells, CD4+ cells isolated from the spleen were differentiated to Th1 and Th2 cells. Subsequent functional analysis showed a significant change in their marker cytokine production. Real-time PCR analysis revealed decreased mRNA levels of Phb1 and Phb2 in both (male and female) Phb1-Ki mice compared with wild-type mice. In summary, findings from Phb1-Ki mice suggest a role of Cys69-linked function in mediating PHB1’s sexually dimorphic immune function in lymphoid tissues, which may in part be mediated through its homologue and heterodimeric partner protein PHB2 because of their multifaceted relationship with sex steroids.

Cell-intrinsic early events involved in different trophic hormone-induced steroidogenesis in their respective steroidogenic cell type are very similar. For example, the activation of the cAMP-PKA signaling pathway in response to trophic hormone stimulation and, subsequently, cholesterol transport to the mitochondria to initiate steroidogenesis is common to them. Recently, we have found that an evolutionarily conserved protein, prohibitin-1 (PHB1), is regulated by Luteinizing Hormone (LH) in murine Leydig cells and plays a role in interconnected cell signaling and mitochondrial steps pertaining to testosterone production. Among the primary steroidogenic tissues, PHB1 expression levels are highest in the adrenal cortex (The Human Protein Atlas); however, its regulation and role in adrenocortical cells are virtually unknown. We investigated the regulation and the role of PHB1 in adrenocortical cells in vitro using human HAC15 and mouse Y-1 cell culture models. It was found that Adrenocorticotrophic Hormone (ACTH) stimulation upregulates PHB1 levels in adrenocortical cells in a time-dependent manner. A similar effect on PHB1 levels was also observed in response to dibutyryl-cAMP stimulation, a cell-permeable analogue of cAMP (the second messenger for ACTH action). Moreover, manipulating PHB1 levels in adrenocortical cells affected mitochondria, lysosomes, and lipid droplet characteristics, modulated phospho-PKA and phospho-ERK1/2 levels, and altered corticosteroid production. This finding suggests that ACTH regulates PHB1 in adrenocortical cells and plays a role in corticosteroid production, which was previously unknown.

Prohibitin (PHB) is an evolutionarily conserved protein capable of modulating the metabolic status of multiple cell types. Phosphorylation of PHB at tyrosine residues regulates signal transduction and intracellular trafficking by hormones, growth factors and antigen-activated immune cells. Our laboratory has developed two transgenic mouse models by overexpressing PHB and a mutated PHB (Y114F-PHB) under the fatty acid-binding protein 4 (Fabp4) gene promoter, which is selectively expressed in adipocytes and monocytic macrophages. Based on discoveries from these two transgenic mouse models named Mito-Ob and mutant-Mito-Ob, I speculate that PHB plays an important role in modulating macrophage polarization and function in part through its Tyr-114 phosphorylation (Chapter 2). Macrophages can polarize into two major phenotypes: proinflammatory (M1) and anti-inflammatory (M2). This functional plasticity of macrophages is heavily influenced by internal and external signals, which alter their survival and output. In this thesis, I examined the role of PHB in the metabolism and immune function of macrophages. The importance of PHB tyrosine phosphorylation in cell signalling is reviewed in Chapter 3. PHB has proven to be a crucial link in endocrine-immune crosstalk. In Chapter 4, the immunometabolic aspect of PHB function is reviewed in the context of Mito-Ob and mutant-Mito-Ob mice based on their metabolic and immune dysregulations. While PHB function was examined in adipocytes during my master’s thesis, its role in macrophages remains unexplored. In Chapter 5, I investigated the role of PHB under the M1/M2 dichotomy in a murine cell line, RAW 264.7, and the Mito-Ob and mutant-Mito-Ob mouse models. PHB’s involvement in the functional plasticity of macrophages is many folds. Overexpression of PHB in macrophages differentially affected cytokine production in M1 and M2 macrophages in vitro. Mutation of the Tyr-114 phosphorylation site in PHB affected ERK and STAT6 signalling, arg

Adrenocortical cells — the corticosteroids-producing cells of the adrenal glands — are rich in cholesterol containing lipid droplets (LDs) and mitochondria, and both are steroidogenically interconnected. The former serves as the precursor substrate for corticosteroids biosynthesis whereas the later represents the site where corticosteroidogenesis begins. The corticosteroidogenesis has been studied extensively; however, a number of fundamental questions related to adrenocorticotrophic hormone (ACTH)-induced and basal steroidogenesis remain unclear, including cholesterol transport to the inner mitochondrial membrane (IMM) where steroidogenesis initiates and the role of intracellular cholesterol pool. Prohibitin-1 (PHB1) is an evolutionarily conserved protein that localizes to the IMM, where it functions as a lipid and protein chaperone. Recently, our laboratory has discovered that PHB1 plays a role in testicular Leydig cell steroidogenesis, involving cholesterol homeostasis and mitochondrial biology pertaining testosterone production. Thus, the present study investigates the role of PHB1 in corticosteroidogenesis (in vitro using human HAC15 and mouse Y-1 cell culture models), as the fundamental aspects of steroidogenesis related to cholesterol handling and mitochondrial biology are similar in different steroidogenic cell types. Manipulation of PHB1 level was found to affects mitochondria, lysosomes and lipid droplets characteristics, as well as corticoid production by adrenocortical cells. Mechanistically, I found that PHB1 modulates PKA and ERK signaling in adrenocortical cells. A consistent finding of PHB1’s role in steroidogenesis in adrenocortical cells from two different species, as well as previously in testosterone producing murine Leydig cells imply that its role in steroidogenesis is likely conserved. Moreover, as the framework of corticosteroidogenesis is built around cholesterol — the precursor substrate for steroid hormones, including its cellular uptake b

The fundamental framework of steroidogenesis across steroidogenic cells is similar, especially the initial mitochondrial steps involving cholesterol translocation across outer mitochondrial membrane (OMM) by the START domain containing proteins (e.g., StAR) and subsequent conversion to pregnenolone by the enzyme P450scc in the inner mitochondrial membrane (IMM). Thus, cholesterol and mitochondria are essential and highly interconnected in steroidogenesis. However, our understanding of this conserved process in steroidogenesis remains limited. Particularly, the transport of cholesterol from the OMM to the IMM. Moreover, growing evidence suggest an important role of autophagy/lipophagy and mitochondrial dynamics in regulating steroidogenic cholesterol homeostasis. However, a potential role the intracellular cholesterol pool in itself in the regulation steroidogenic events remains unexplored. I investigated an unexpected testicular phenotype of two transgenic mouse models and found that the male transgenic mice that expressed a mutant form of a mitochondrial protein prohibitin-1 (PHB1Tyr114Phe) from the Fabp-4 gene promoter displayed smaller testes, higher testosterone levels and lower gonadotropin levels as compared with age matched PHB-1 overexpressing and wild-type mice. Additionally, I found StAR and P450scc as interacting partners of PHB1 in Leydig cell mitochondria. Further analysis of testis and Leydig cells from the transgenic mice revealed that PHB1 plays a regulatory role in coordinating cell signaling, cholesterol homeostasis, and mitochondrial biology pertaining to steroidogenesis. Moreover, I tested the hypothesis that intracellular cholesterol pool in steroidogenic cells plays a role in regulating cell-intrinsic factors and events pertaining steroidogenesis. I found that the depletion of intracellular cholesterol pool in steroidogenic cells (i.e., MA-10, Y-1 and BeWo cells) induces autophagy, affects mitochondrial dynamics, and upregulates steroidogenic f

Many molecular bases for the long-term adaptive changes in cells remain unknown. Alternative splicing contributes dramatically to protein diversity and allows for the fine-tuning of gene functions in response to changing extracellular stimulation. It’s a flexible process that could be spatiotemporally and dynamically regulated in response to various extracellular stimuli by the combined effects of specific RNA elements, cis- and trans-acting factors, epigenetic modifications as well as other regulatory factors. The effect of repeated stimulations on gene expression can often be distinct from that of a single stimulation, like in sustained regular exercises or long-term drug abuse. However, it remains unclear how splice variants are controlled in this process, and whether DNA methylation or MeCP2 (Methyl-CpG Binding Protein 2) regulate this potential process. Here, by a depolarization-splicing system, it shows that while the majority of exons kept their homeostatic levels after repeated stimulations, a small group of synaptic exons are indeed adaptively spliced in a significantly different way from or even opposite to that by a single treatment upon membrane depolarization. For further exploration, I established an exon DNA methylation-splicing reporter assay system to conveniently examine the methylation effect on splicing by mutagenesis. In combination with a pair-wise genome/transcriptome-wide analysis, it shows that the adaptively spliced exons are controlled by DNA methylation. More interestingly, a group of adaptive synaptic exons is aberrantly spliced upon DNA methylation change in rat GH3 pituitary cells or due to mutation of the mC- or splicing factor-binding domains of the MeCP2 in patients with Rett syndrome, an autism spectrum disorder. In this thesis, the data established the role of DNA methylation in the adaptive splicing of synaptic exons in response to repeated stimulations by cellular activities and demonstrated similar changes in MeCP2-mutated Rett

The emergence of exons and introns in eukaryotes has increased the potential of gene products and their functional diversity to a greater extent than in prokaryotes. In humans, the transcriptome has the highest percentage of multi-exon genes that contain at least one alternatively spliced exon. At the exon level, however, it is the constitutive exons that have been observed more often in gene transcripts. This trend has been shifting with the increasing numbers of alternative exons per gene. Here, we estimate the differentially used exons (DEXs) of human RNA transcripts at a saturation level among 1,540 pairs of 56 nominally normal tissues with DEXs comprising at least 72% of the human exome. Our result has shifted the paradigm of exon usage from mainly constitutive to differentially used exons in the human transcriptome, suggesting a more diverse and dynamic transcript repertoire of the human transcriptome than previously reported. We used this comprehensive catalogue of DEXs of the normal transcriptome as background for further analysis against the cancer transcriptomes. We demonstrated that 99% of the aberrantly processed exons of adenocarcinoma also exist in the normal transcriptome blueprint of DEXs showing similar processing pattern as normal in other non-cancerous tissues. A small group of authentic ‘cancer-specific’ exons that were found only in cancer patients harboring specific gene mutations, were not found in any of the normal tissues. Thus, our results also reveal the ectopic expression of nearly all ‘cancer-specific’ exons of adenocarcinoma compared to the corresponding normal tissue of origin. This will help selection for authentic ‘cancer-specific’ target exons for diagnostic or therapeutic purposes.

Hyaluronic acid (HA) is abundantly present in the extracellular matrix (ECM) and reported to have a functional role during embryonic development. Among ECM components, HA has one of the fastest rates of turnover and is facilitated by hyaluronidases (HYALs). HYAL1 and HYAL2 are the predominant HYALs associated with the HA catabolism in somatic tissues. HYAL2 being a GPI-anchored cell-surface hyaluronidase is presumed to generate HA fragments (10-20 kDa) from large-sized extracellular HA (>200 kDa) followed by further degradation in the lysosomes into tetrasaccharides. Hyal2 knockout (KO) mice displayed craniofacial abnormalities and severe pre-weaning lethality. High frequency ultrasound of Hyal2 KO mouse hearts revealed atrial dilation and 50 % of Hyal2 KO mice exhibited a triatrial heart (cor triatriatum). Two separate mutations- K148R and P250L in HYAL2 were found to be associated with syndromic cleft palate. Recently, whole exome sequencing revealed seven new HYAL2 variants and we hypothesized that these gene variants interfere with the cellular role of HYAL2 by affecting its stability and cell-surface localization. Very low or a complete absence of expression were observed from the variants and the cell-surface localization of HYAL2 were severely compromised. Our biochemical analysis confirms that these seven variants are novel HYAL2 mutations. Levels of HA during different stages of embryonic development are finely regulated. The action of HYAL2 towards HA removal can be explained by the excessive accumulation of HA in Hyal2 KO mouse tissues. But, assaying the activity of HYAL2 directly has been a challenge in the field likely due to the lack of enough information on its mechanism of action. We speculate that HYAL2’s activity is regulated to allow its activation only at specific times. Searching for possible interacting protein partners of HYAL2 can provide mechanistic insight into the HYAL2 regulation. We employed proximity labeling system to report a library

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic idiopathic inflammatory bowel diseases (IBD) with an estimated prevalence in Canada of around 15/100000 persons. Biological agents are expensive, require indefinite use to ensure a sustained response, and raise concerns about infections and other complications thus limiting enthusiasm for introducing them earlier in the treatment for IBD. The essential cells in the innate immune system that coordinates the inflammatory process are the macrophages (MØ), where MØs have been demonstrated to be increased in areas of inflammation in IBD. MØs are a heterogeneous population of two different phenotypes: group 1: “inflammatory, classically activated MØs: M1” and alternative activated MØs: M2. MØs polarization and plasticity depend on specific combinations of cytokines and peptides within the microenvironment. Mucosal changes in IBD are characterized by inflammation and are linked to hyperplasia in the chromogranin-A (CHGA)-producing enterochromaffin (EC) cells. CHGA cleavages generate several biologically active peptides, including chromofungin (CHGA47-66 [CFG]). Our lab demonstrated that CFG is an essential peptide in MØs regulation. Genome-wide associated studies in IBD have revealed that in IBD and within the epithelium, endoplasmic reticulum (ER) stress and intestinal epithelial apoptosis figure strikingly. A p53-apoptotic pathway mediates intestinal epithelial apoptosis through the activation of the p53-upregulated modulator of apoptosis (PUMA), Bcl-2 associated-X protein (BAX), Bcl-2 associated death promoter (BAD), Bcl-2 antagonist/killer-1 (BAK1), Caspase-3 and Caspase-8 proteins. Polymorphisms of X-box binding protein 1 (XBP1) gene, a main UPR signalling protein (GRP78), that develop enteritis and subsequent apoptotic cell death and is associated with an increased risk of IBD, The deletion of CHGA leads to an increase in GRP78 expression in diabetes. Currently, there is a gap of knowledge in understanding