Faculty Bibliography

GPCRs transform extracellular stimuli into a physiological response by activating an intracellular signaling cascade initiated via binding to G proteins. Orphan G protein-coupled receptors (GPCRs) hold the potential to pave the way for development of new, innovative therapeutic strategies. In this review we will introduce G protein-coupled receptor 143 (GPR143), an enigmatic receptor in terms of classification within the GPCR superfamily and localization. GPR143 has not been assigned to any of the GPCR families due to the lack of common structural motifs. Hence we will describe the most important motifs of classes A and B and compare them to the protein sequence of GPR143. While a precise function for the receptor has yet to be determined, the protein is expressed abundantly in pigment producing cells. Many GPR143 mutations cause X-linked Ocular Albinism Type 1 (OA1, Nettleship-Falls OA), which results in hypopigmentation of the eyes and loss of visual acuity due to disrupted visual system development and function. In pigment cells of the skin, loss of functional GPR143 results in abnormally large melanosomes (organelles in which pigment is produced). Studies have shown that the receptor is localized internally, including at the melanosomal membrane, where it may function to regulate melanosome size and/or facilitate protein trafficking to the melanosome through the endolysosomal system. Numerous additional roles have been proposed for GPR143 in determining cancer predisposition, regulation of blood pressure, development of macular degeneration and signaling in the brain, which we will briefly describe as well as potential ligands that have been identified. Furthermore, GPR143 is a promiscuous receptor that has been shown to interact with multiple other melanosomal proteins and GPCRs, which strongly suggests that this orphan receptor is likely involved in many different physiological actions.

Acyl-CoA synthetase 1 (ACSL1) is an enzyme that converts fatty acids to acyl-CoA-derivatives for lipid catabolism and lipid synthesis in general and can provide substrates for the production of mediators of inflammation in monocytes and macrophages. Acsl1 expression is increased by hyperglycemia and inflammatory stimuli in monocytes and macrophages, and promotes the pro-atherosclerotic effects of diabetes in mice. Yet, surprisingly little is known about the mechanisms underlying Acsl1 transcriptional regulation. Here we demonstrate that the glucose-sensing transcription factor, Carbohydrate Response Element Binding Protein (CHREBP), is a regulator of the expression of Acsl1 mRNA by high glucose in mouse bone marrow-derived macrophages (BMDMs). In addition, we show that inflammatory stimulation of BMDMs with lipopolysaccharide (LPS) increases Acsl1 mRNA via the transcription factor, NF-kappa B. LPS treatment also increases ACSL1 protein abundance and localization to membranes where it can exert its activity. Using an Acsl1 reporter gene containing the promoter and an upstream regulatory region, which has multiple conserved CHREBP and NF-kappa B (p65/RELA) binding sites, we found increased Acsl1 promoter activity upon CHREBP and p65/RELA expression. We also show that CHREBP and p65/RELA occupy the Acsl1 promoter in BMDMs. In primary human monocytes cultured in high glucose versus normal glucose, ACSL1 mRNA expression was elevated by high glucose and further enhanced by LPS treatment. Our findings demonstrate that CHREBP and NF-kappa B control Acsl1 expression under hyperglycemic and inflammatory conditions.

Wildtype or mutant proteins expressed beyond the capacity of a cell's protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes, and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.

As obligate intracellular parasites with reduced genomes, microsporidia must infect host cells in order to replicate and cause disease. They can initiate infection by utilizing a harpoon-like invasion organelle called the polar tube (PT). The PT is both visually and functionally a striking organelle and is a characteristic feature of the microsporidian phylum. Outside the host, microsporidia exist as transmissible, single-celled spores. Inside each spore, the PT is arranged as a tight coil. Upon germination, the PT undergoes a large conformational change into a long, linear tube and acts as a tunnel for the delivery of infectious cargo from the spore to a host cell. The firing process is extremely rapid, occurring on a millisecond timescale, and the emergent tube may be as long as 20 times the size of the spore body. In this chapter, we discuss what is known about the structure of the PT, the mechanics of the PT firing process, and how it enables movement of material from the spore body.

Troxerutin (TXR) is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit the antiarthritic properties of TXR using an adjuvant-induced arthritic (AIA) rat model. AIA-induced rats showed the highest arthritis score at the disease onset and by oral administration of TXR (50, 100, and 200 mg/kg body weight), reduced to basal level in a dose-dependent manner. Isobaric tags for relative and absolute quantitative (iTRAQ) proteomics tool were employed to identify deregulated joint homogenate proteins in AIA and TXR-treated rats to decipher the probable mechanism of TXR action in arthritis. iTRAQ analysis identified a set of 434 proteins with 65 deregulated proteins (log₂ case/control≥1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from the healthy ones were validated using Western blot analysis. The Western blot data corroborated proteomics findings. In silico protein-protein interaction study of tissue-proteome revealed that complement component 9 (C9), the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, get perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an antiarthritic agent in the AIA model and after additional investigation, its arthritic ameliorating properties could be exploited for clinical usability.

Polymorphisms in the Apolipoprotein L1 (APOL1) gene are common in ancestrally African populations, and associate with kidney injury and cardiovascular disease. These risk variants (RV) provide an advantage in resisting Trypanosoma brucei, the causal agent of African trypanosomiasis, and are largely absent from non-African genomes. Clinical associations between the APOL1 high risk genotype (HRG) and disease are stronger in those with comorbid infectious or immune disease. To understand the interaction between cytokine exposure and APOL1 cytotoxicity, we established human umbilical vein endothelial cell (HUVEC) cultures representing each APOL1 genotype. Untreated HUVECs were compared to IFNÉ£-exposed; and APOL1 expression, mitochondrial function, lysosome integrity, and autophagic flux were measured. IFNÉ£ increased median APOL1 expression across all genotypes 22.1 (8.3 to 29.8) fold (p=0.02). Compared to zero risk variant-carrying HUVECs (0RV), HUVECs carrying 2 risk variant copies (2RV) showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosomal compartment, and an accumulation of autophagosomes suggesting a defect in autophagic flux. Upon blocking autophagy with non-selective lysosome inhibitor, hydroxychloroquine, autophagosome accumulation between 0RV HUVECs and untreated 2RV HUVECs was similar, implicating lysosomal dysfunction in the HRG-associated autophagy defect. Compared to 0RV and 2RV HUVECs, HUVECs carrying 1 risk variant copy (1RV) demonstrated intermediate mitochondrial respiration and autophagic flux phenotypes, which were exacerbated with IFNÉ£ exposure. Taken together, our data reveal that IFNÉ£ induces APOL1 expression, and that each additional RV associates with mitochondrial dysfunction and autophagy inhibition. IFNÉ£ amplifies this phenotype even in 1RV HUVECs, representing the first description of APOL1 pathobiology in variant heterozygous cell cultures.

OBJECTIVE/UNASSIGNED:The present study aimed to enhance understanding of continuity and stability of positive parenting of infants, across age and different settings in women with a history of depression who are at elevated risk for postpartum depression. DESIGN/UNASSIGNED:= 103) with a history of major depression and their infants were observed during 5-min play and feeding interactions when their infants were 3, 6, and 12 months of age. Summary scores representing mothers' positive parenting were computed separately for each age and context based on ratings of five parenting behaviors. Mothers' depressive symptom levels were assessed at each infant age. RESULTS/UNASSIGNED:Continuity (consistency of level) and stability (consistency of rank order) were assessed across age and context at both the group and individual level. Across-age analyses revealed continuity in the play context and discontinuity in the feeding context, albeit only at the group level, as well as weak to moderate stability. Across-context analyses revealed higher positive parenting scores in play than feeding at all time points as well as weak to moderate stability. Variations in positive parenting across age and context were independent of mothers' postpartum depressive symptom levels. CONCLUSIONS/UNASSIGNED:Findings based on normative samples may not generalize to women with a history of depression, who may benefit from interventions aimed at enhancing their positive parenting over the course of infancy, regardless of postpartum depressive symptom level. Results also underscore the importance of assessing parenting at multiple age points and across varying contexts.

Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these "orphan" (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important for maintaining cell structure, but strong fixation and vigorous dehydration (as it is routine for EM) can alter protein structure, thus impairing antigen-antibody binding. Here, we implemented a method to combine pre-embedding immunolabeling (pre-embedding) with serial block-face scanning electron microscopy (SBF-SEM). We utilized a murine model of cardiomyocyte-specific, Tamoxifen (TAM) activated knockout of PKP2. Adult hearts were harvested 14 days post-TAM, at this time hearts present a phenotype of concealed ARVC (i.e., an arrhythmogenic phenotype but no overt structural disease). Thick (200 µm) vibratome slices were immunolabelled for Cx43 and treated with nanogold or FluoroNanogold, coupled with a silver enhancement. Left or right ventricular free walls were dissected and three-dimensional (3D) localization of Cx43 in cardiac muscle was performed using SBF-SEM. Reconstructed images allowed us to visualize the entire length of gap junction plaques, seen as two parallel, closely packed strings of Cx43-immunoreactive beads at the intercalated disc. In contrast, in PKP2-deficient hearts we observed bulging of the intercellular space, and entire areas where only one of the two strings could be observed, indicating the presence of orphan Cx43. We conclude that pre-embedding and SBF-SEM allowed visualization of cardiac Cx43 plaques in their native environment, providing for the first time a visual complement of functional data indicating the presence of orphan Cx43 hemichannels resulting from loss of desmosomal integrity in the heart.

Mammalian spermatogenesis is associated with the transient appearance of condensed mitochondria, a singularity of germ cells with unknown function. Using proteomic analysis, respirometry, and electron microscopy with tomography, we studied the development of condensed mitochondria. Condensed mitochondria arose from orthodox mitochondria during meiosis by progressive contraction of the matrix space, which was accompanied by an initial expansion and a subsequent reduction of the surface area of the inner membrane. Compared to orthodox mitochondria, condensed mitochondria respired more actively, had a higher concentration of respiratory enzymes and supercomplexes, and contained more proteins involved in protein import and expression. After the completion of meiosis, the abundance of condensed mitochondria declined, which coincided with the onset of the biogenesis of acrosomes. Immuno-electron microscopy and the analysis of sub-cellular fractions suggested that condensed mitochondria or their fragments were translocated into the lumen of the acrosome. Thus, it seems condensed mitochondria are formed from orthodox mitochondria by extensive transformations in order to support the formation of the acrosomal matrix.