The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC-independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation. / Ph. D.

No / Chemotherapeutic agents induce p53-dependent apoptosis in the hair follicle (HF) resulting in hair loss, a common side effect of cancer therapy. Here, we show that Fas as a p53 target plays important role in the HF response to cyclophosphamide. Specifically, we demonstrate that Fas is up-regulated in HF keratinocytes after cyclophosphamide treatment, Fas ligand-neutralizing antibody partially inhibits HF response to cyclophosphamide in wild-type mice, and Fas knockout mice show significant retardation of cyclophosphamide-induced HF involution associated with reduced Fas-associated death domain and caspase-8 expression. These data raise a possibility to explore blockade of Fas signaling as a part of complex local therapy for inhibiting keratinocyte apoptosis and hair loss induced by chemotherapy.

Damage to the vasculature is a common occurrence in diabetes mellitus. At the cellular level, dysfunction of vascular endothelial cells is often associated with diabetic conditions. Multiple agents maintain the endothelium, including vascular endothelial growth factor (VEGF), an endothelial cell mitogen/survival factor, and insulin, which has anti-apoptotic effects on endothelial cells in addition to regulating glucose homeostasis. Insulin and VEGF, upon activating their respective tyrosine kinase receptors, can engage the PI3-kinase/Akt, MAPK, and PLC-γ/PKC pathways. Thus, crosstalk between VEGF and insulin signaling may occur at numerous points. Our objectives were twofold: 1) to characterize the combined effects of insulin and VEGF on downstream elements, and 2) to determine the ability of signaling intermediates principally associated with either insulin or VEGF signaling to interact directly. After treatment with VEGF, insulin, or both, cells expressing both VEGF receptor-2 (KDR) and the insulin receptor were immunoprecipitated for total Akt and PLC-γ. Isolates from cells stimulated with both ligands demonstrated activation of PLC-γ and Akt that was less than additive over fifteen minutes. Conversely, cells pretreated with advanced glycation end products showed increased Akt phosphorylation. The effect of insulin on VEGF bioactivity was also measured by PLC-γ-mediated hydrolysis of phosphatidylinositol. These studies suggested suppressed VEGF activity in the presence of insulin. To examine direct signaling interactions, recombinant reagents capable of selective binding (via SH2 domains) to phosphorylated receptors were generated. Overall results showed relatively unaffected VEGF activity in the presence of insulin; however, this relationship is likely altered within the diabetic state. / Master of Science

The phylum of Chlamydiota is composed of gram negative obligate intracellular bacteria that live as symbionts of diverse eukaryotes, from protists to animals and humans. Members of the phylum can be split into two groups: the environmental Chlamydia, which includes symbionts of amoeba, and the pathogenic Chlamydia, which includes species infecting animals, birds, and humans and includes Chlamydia trachomatis the leading cause of reportable, bacterial sexually transmitted infections and the ocular infection, trachoma. The characterized phylum members undergo a biphasic developmental cycle alternating between the infectious elementary body (EB) and the replicative reticulate body (RB), with each form having distinct morphological and physiological properties. Differentiation between these forms occurs within a host cell membrane-derived vacuole termed the inclusion. The molecular mechanisms governing and executing bacterial development and RB growth remain unclear. The essentiality and uniqueness of development makes it a prime target for the development of novel, chlamydial-specific therapeutics. Reductive evolution has resulted in the loss of or fragmentation of numerous metabolic pathways, particularly in the pathogenic Chlamydia (~1 Mbp genome) as compared to the environmental Chlamydia (~2.5 Mbp). We hypothesize that the bacterium senses environmental changes (host cytoplasm) to ensure that development and growth coincide with host cell energy and metabolite levels. We predict that an encoded partner switching mechanism (PSM) plays a key role in: 1) regulation of growth by acting as a molecular throttle through regulation of the housekeeping sigma factor, and 2) differentiation by impacting the composition of the sigma factor pool allowing for transcriptional changes needed for developmental transitions. We also predict that PSM regulation occurs through sensing of nucleotide triphosphates, TCA-cycle intermediates, metal concentrations, and redox. Canonical PSMs have a PP2C-type sensor phosphatase (SP), an anti-sigma factor (ASF, serine kinase), an anti-anti-sigma factor (AASF, substrate of the SP and ASF) and a stress-response related alternative sigma factor. The PSM in pathogenic Chlamydia is atypical, and despite its reduced genome, is comprised of two SPs (RsbU which responds to α-ketoglutarate and CTL0852), two AASFs (RsbV1 and RsbV2), one ASF (RsbW), and, unusually, the ASF regulates the availability of the “housekeeping” sigma factor, σ66. To test our hypotheses, we first constructed and purified a variety of amino acid point mutants of the two AASFs, ASF, and the SP for in vitro analyses. Kinase and phosphatase activity towards RsbV1/V2 was measured in the presence of different metals, phosphate donors, and pH and redox conditions. Phos-tag acrylamide gels were used to assess protein phosphorylation status. We discovered that metalation impacts enzyme activity and the substrate specificity of RsbU, and that RsbW can use multiple phosphate donors. Prior work, and our data, found that RsbW and RsbU have higher enzymatic activity towards RsbV1 than RsbV2, leading us to explore the importance of RsbV2 in chlamydial biology. Genome gazing revealed that environmental Chlamydia possess a single AASF, and bioinformatic analyses support that it is more similar to RsbV2 than RsbV1 suggesting that the pathogenic Chlamydia gained RsbV1. Comparing the biochemical features of the two AASFs provides potential reasons for the different enzyme affinities. To flesh out the in vivo importance of each AASF, we characterized bacterial growth, infectious progeny production, and the levels of RsbV1/V2 in a cell culture infection model using a collection of C. trachomatis L2 rsbV1 null or rsbV2 knockdown strains. We also overexpressed the AASFs in strains grown with different glucose levels. Note that C. trachomatis is an auxotroph for glucose-6-phosphate. In normal chlamydial culture glucose medium levels, the rsbV1 null strain showed an ~1 log reduction in infectious progeny numbers while the rsbV2 knockdown or AASF overexpression strains had no defects. We also observed that the rsbV1 null strain has a developmental delay and exhibits growth differences in response to glucose levels, i.e. a functional PSM seems to set a “growth cap” in response to different glucose availability. Immunoblotting analysis of RsbV1/V2 demonstrated the presence of both proteins throughout development, and protein levels remained the same in low or high glucose levels and in the wild type or rsbV1 null strains (measuring RsbV2 only for the RsbV1 null strain). These results tell us that the AASF levels have minimal impact on chlamydial biology, suggesting that phosphorylation status is key to regulation. To assess phosphorylation, we used protein pulldown assays and Phos-tag gels to assess RsbV1 and RsbV2 phosphorylation during development. Both RsbV1 and RsbV2 were phosphorylated during the EB stage, which is similar to our prior results using Chlamydia caviae. In conjunction with the in vivo phosphorylation data, we hypothesize that stage-dependent inhibition of AASF/RsbW interactions frees RsbW to sequester σ66. Reduced pools of σ66 would promote RB-EB conversion through increased RNAP binding to the late gene sigma factors σ54 and σ28. Supporting this model, overexpression of a non-phosphorylatable RsbV2 S55A mutant (an RsbW “trap”), but not overexpression of RsbV1 S56A, resulted in a 3 log reduction in infectious progeny production without gross changes in inclusion morphology or bacterial numbers, while causing a reduction in σ54 and σ28 regulated EB-specific proteins and inhibition of RB-EB transition shown via transmission electron microscopy. As an alternative approach to assess the consequence of reduced “free” RsbW, we used a CRISPRi knockdown system targeting rsbW and observed a reduction in infectious progeny production under some conditions, which is consistent with the RsbV2 S55A expression strain results. The rsbW CRISPRi-associated phenotype was weaker than the RsbV2 S55A phenotype. As bacterial redox status changes throughout development (RBs are reduced and EBs are oxidized), we also assessed whether the cysteine-rich proteins RsbV2 and RsbW were redox responsive. In parallel to the unique AASF expansion in the pathogenic Chlamydia, RsbV2 in the pathogenic Chlamydia has a CXCC motif that is not found in the RsbV homolog in the environmental Chlamydia. Our in vitro studies found that, under oxidizing conditions, RsbV2 is dimerized, and the dimer form inhibits phosphorylation of RsbV2 by RsbW. We predict that retention of RsbV2 after RsbV1 acquisition has been selected for, in part, owing to a unique redox-sensing role compared to RsbV1 and that the presence of two AASFs enables more sensitive tuning of growth and development in response to metabolite levels. The different phenotypes when overexpressing non-phosphorylatable RsbV1 and RsbV2 also hints at a potential non-PSM or expanded PSM role for RsbV1. The in vitro redox findings need to be further explored in an in vivo model. Collectively, we think the expansion of the PSM, in addition to other gene gain events, facilitated infection of multi-cellular organisms. Additionally, our data support that the PSM regulates growth/cell differentiation in response to energy/nutrients, and that redox levels and biochemical features of RsbV1 and RsbV2 govern PSM-component interactions. As disruption of normal PSM function significantly reduces production of infectious progeny, compounds targeting the PSM components could serve as novel, narrow spectrum inhibitors.

No description available.

No description available.

published_or_final_version / Pathology / Master / Master of Philosophy

The influenza A virus non-structural (NS1) protein is multifunctional, and during virus-infection NS1 interacts with several factors in order to manipulate host-cell processes. This study reports that NS1 binds directly to p85β, a regulatory subunit of phosphoinositide 3-kinase (PI3K), but not to the related p85α. Expression of NS1 was sufficient to activate PI3K and cause the phosphorylation of a downstream mediator of PI3K signalling, Akt. However, in virus-infected MDCK cells, the kinetics of Akt phosphorylation did not correlate with NS1 expression, and suggested that negative regulation of this signalling pathway occurs subsequent to ~8h post-infection. Mapping studies showed that the NS1:p85β interaction is primarily mediated by the NS1 C-terminal domain and the p85β inter-SH2 (Src homology 2) domain. Additionally, the highly conserved tyrosine at residue 89 (Y89) of NS1 was found to be important for binding and activating PI3K in a phosphorylation-independent manner. The inter-SH2 domain of p85β is a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. As NS1 does not displace p110 from the inter-SH2 domain, a model is proposed whereby NS1 forms an active heterotrimeric complex with PI3K, and disrupts the ability of p85β to control p110 function. Biological studies revealed that a mutant influenza A virus (Udorn/72) expressing NS1 with phenylalanine substituted for tyrosine-89 (Y89F) exhibited a small-plaque phenotype, and grew more slowly in MDCK cells than wild-type virus. Unexpectedly, another mutant influenza A virus strain (WSN/33) expressing NS1-Y89F was not attenuated in MDCK cells, yet appeared to be less pathogenic than wild-type in vivo. Overall, these data indicate a role for NS1-mediated PI3K activation in efficient influenza A virus replication. The potential application of this work to the design of novel anti-influenza drugs and vaccine production is discussed.

Les prions sont des agrégats amyloïdes infectieux. Le prion [Het-s] de Podospora anserina est un des prions le mieux caractérisé. Le prion [Het-s] est impliqué dans l’incompatibilité végétative – un processus biologique qui a lieu au cours des anastomoses entre des souches génétiquement différentes. Quand une souche [Het-s] fusionne avec une souche exprimant l’allèle alternatif du gène het-s – l’allèle het-S – une réaction de mort cellulaire programmée est déclenchée. Les deux protéines diffèrent de 13 acides aminés et partagent une architecture en deux domaines ; un domaine globulaire en N-terminal nommé HeLo et un domaine PFD (Prion Forming Domain) en C-terminal. Il a été établi qu’en présence des fibres amyloïdes de [Het-s], la protéine HET-S agit en ‘pore-forming’ toxine : la transconformation du PFD de HET-S par les fibres amyloïdes du [Het-s] active le domaine HeLo de HET-S et entraîne la mort cellulaire. Afin de mieux caractériser les propriétés du repliement β-solénoïde du prion [Het-s], nous avons entrepris l’exploration in vivo des relations structure-fonction de ce repliement par une approche d’alanine scanning. Au cours de nos recherches pour des homologues de HET-S/s, nous avons identifié un partenaire fonctionnel de HET-S – une protéine appelée NWD2. NWD2 est une protéine STAND et partage une séquence homologue (3-23) au PFD de HET-S/s. Les protéines STAND, après la reconnaissance d’un ligand, forment des plateformes oligomériques pour transduire le signal. Des analyses génomiques in silico réalisées dans plusieurs génomes fongiques nous ont amené à proposer que la transduction du signal via une protéine STAND à repliement amyloïde est un mécanisme ancien et conservé chez les champignons. Dans ce contexte nous avons identifié deux nouveaux motifs PFD putatifs – σ et PP. En soumettant à l’épreuve notre hypothèse, nous avons d’abord démontré que NWD2 interagit avec HET-S/s en fonction d’un ligand spécifique in vivo et l’interaction est dépendante de la séquence NWD2(3-23) homologue au PFD de HET-S/s. Nous avons ensuite exploré le motif PP associé à un domaine HeLo-like (HELL) dans le génome de Chaetomium globosum. En démontrant la nature amyloïde et prion-like du motif PP ainsi que l’analogie fonctionnelle entre ce motif et le PFD de HET-S/s in vivo nous avons apporté des arguments supplémentaires en faveur de l’implication des repliements amyloïdes dans la transduction du signal chez les champignons filamenteux. / Prions are infectious amyloid aggregates. Podospora anserina’s [Het-s] is one of the best characterized fungal prions with a remarkably high prevalence in wild populations. [Het-s] functions in vegetative incompatibility - a biological process occurring during anastomosis between two genetically incompatible strains. When an [Het-s] prion infected strain fuses with a strain expressing the alternative allelic variant of the het-s locus – het-S – a cell death reaction of the heterokaryon occurs. Differing by 13 amino acids both proteins shares two domain architecture; a globular N-terminal domain called HeLo and a C-terminal Prion Forming Domain (PFD). It has been demonstrated that in presence of [Het-s] amyloid fibers HET-S turns into a pore-forming toxin: transconformation of the HET-S PFD by [Het-s] fibers triggers the refolding of the HET-S HeLo domain, inducing the cell death reaction. In an attempt to better characterize the conserved features of the [Het-s] β-solenoid fold we have used a mutational alanine scanning approach and explored in vivo the existing relations between structure and prion functions of [Het-s]. During our quest for new distant homologues of HET-S/s, we have identified a functional partner of HET-S toxin called NWD2. NWD2 is a STAND protein and shares a homology sequence (3-23) in the HET-S/s PFD. STAND proteins form signal transducing hubs through oligomerization upon ligand recognition. Several in silico analysis in various fungal genomes led us to propose that signal transduction via a STAND protein using an amyloid prion-like fold is a general widespread mechanism in fungi. In that context, we have proposed two novel putative PFD motifs called σ and PP. Testing experimentally our hypothesis, we have first demonstrated that NWD2 interacts with HET-S/s upon ligand recognition in vivo and the interaction is dependant of the NWD2(3-23) region. We have then explored the newly identified putative prion domain PP, associated to a Helo-like domain (termed HELL) from the filamentous fungus Chaetomium globosum. By demonstrating the amyloid, prion-like nature of the PP motif and the functional analogy between PP and HET-S/s PFD domain in vivo, we expose further evidences supporting the implication of amyloid folds in signal transduction in filamentous fungi.

Hemoproteins represent very important components of many living organisms. Participation in the processes of oxygen transport and storage, electron transport or enzymatic catalysis of reactions involving oxygen or hydrogen peroxide are commonly known functions of hemoproteins. Recently, there has been discovered a new group of hemoproteins. The main feature of this new group of proteins is their ability to detect changes in heme concentration (heme-responsive proteins) or changes in diatomic gas concentration (gas-responsive heme-containing sensor proteins) in their vicinity. Detection of these concentration changes generates signals that induce structural changes of the respective sensor proteins. Finally, the structural changes of the respective sensor proteins affect their functions or activities. The subject of this diploma thesis is the preparation and characterization of the eukaryotic heme sensor Bach1. We especially focused on the ability of Bach1 to bind heme molecules and on the comparison of various Bach1 properties in its apoform and holoform. Determination of the exact amount of heme molecules that specifically interact with heme sensor Bach1 represents very important part of this thesis. We also studied the effect of different redox states of heme iron and the presence of interaction...