Mountain Scholar :: Browsing by Author "Di Pietro, Santiago, committee member"

Browsing by Author "Di Pietro, Santiago, committee member"

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Open Access
5-hydroxymethylcytosine and endonuclease G as regulators of homologous recombination
(Colorado State University. Libraries, 2017) Vander Zanden, Crystal M., author; Ho, P. Shing, advisor; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Fisk, Nick, committee member
Homologous recombination (HR) is a necessary biological process for all living organisms, and it is especially important for repairing damaged DNA. Improper HR results in DNA damage-related diseases, notably increased likelihood of cancer when HR regulators, such as the human BRCA1 gene, are impaired. HR is also a tool for biotechnology, giving scientists the power to easily delete or mutate genes and study the effects of those modifications. Recently, the epigenetically modified nucleotide 5-hydroxymethylcytosine (5hmC) was found to regulate vertebrate HR via interaction with the protein endonuclease G (EndoG). In this dissertation, I use biochemical/biophysical methods to elucidate the interaction between 5hmC and EndoG, thus working towards understanding their roles as regulators of recombination. I find that 5hmC forms a unique hydrogen bond to stabilize Holliday junctions, the four-stranded DNA intermediate in HR. 5hmC also induces a global structure change to the junction, increasing protein access to the junction crossover and providing potential for either direct or indirect readout of 5hmC. Further connecting EndoG with recombination, we present the first evidence that EndoG preferentially binds and cleaves Holliday junction DNA, implicating a role for EndoG as a resolvase. I demonstrate that EndoG recognizes 5hmC in the junction context and observe unique cleavage products from EndoG interaction with 5hmC-junctions. These results suggest that EndoG may have a previously unrecognized junction resolvase function and, in this way, play a more direct role in recombination than simply creating double-stranded breaks in duplex DNA to initiate the HR mechanism. Finally, I present a unique structural feature of vertebrate EndoG that we hypothesize is the basis for 5hmC recognition. I present the structure of mouse EndoG and propose that a two amino acid deletion, conserved in vertebrate EndoG sequences, is associated with unraveling of an α-helix. This structural perturbation positions amino acid side chains to confer 5hmC-sensing ability to all vertebrate EndoG. I expect that these deletion mutations and resulting structural effects co-evolved with the appearance of 5hmC in vertebrate genomes to give EndoG an additional function of recognizing 5hmC in the cell. Overall this work is building onto the understanding of 5hmC and EndoG as markers and regulators of recombination.
Open Access
Androgen signaling in the placenta
(Colorado State University. Libraries, 2014) Cleys, Ellane Rachael, author; Bouma, Gerrit, advisor; Clay, Colin, advisor; Tobet, Stuart, committee member; Di Pietro, Santiago, committee member
Placental estrogen signaling is known to regulate placental trophoblast function and differentiation. However, the role of placental androgen signaling has never been investigated, despite the rise of maternal serum androgens throughout gestation. Recent findings have shown increased maternal serum androgen in patients with the placental induced disorder preeclampsia. Preeclampsia, a maternal hypertension and proteinuria condition instigated by insufficient trophoblast differentiation and invasion into maternal spiral arteries, is also associated with increased placental expression of androgen receptor and an increased risk of incidence in patients with polymorphisms in androgen receptor that decrease androgen signaling. These findings suggest a crucial role for placental androgen signaling. Moreover, research investigating androgen's role in cancer progression has shown that many androgen responsive genes regulate cell proliferation, differentiation to invasive phenotypes, and tissue vascularization, all processes necessary for normal placental development. Androgen signaling in tumor tissues is further regulated by androgen receptor complexes with histone lysine demethylases. These complexes are recruited to androgen response elements in DNA and dynamically regulate histone tail modifications for transcription initiation. This led us to the overall hypothesis that (1) androgen signaling in trophoblast cells is important for placental development, and (2) androgen receptor complexes with histone lysine demethylases in the placenta to regulate vascularization, growth and invasion factors in trophoblast cells. To test this hypothesis, we utilized a prenatal androgenization ewe model as well as human first trimester placental samples and immortalized human trophoblast cell lines. Using the prenatal androgenized ewe model, we report for the first time expression of histone lysine demethylases in the placenta. Furthermore, we showed androgen receptor complexes with histone lysine demethylases and is recruited to an androgen response elements in the 5'untranslated flanking sequence of vascular endothelial growth factor in the sheep placenta. We also report that histone lysine demethylase are present in human first trimester syncytiotrophoblast and complex with androgen receptor in immortalized trophoblasts. Additionally, we demonstrated that androgen receptor complexes with histone lysine demethylases are also present in choriocarcinoma ACH-3P and BeWo cells. Dihydrotestosterone treatment in these cells led to down-regulation of androgen responsive genes, specifically KDM3A and MMP2. Inhibition of androgen receptor through flutamide treatment altered mRNA levels for genes regulating vascularization, including HIF1α, PPARα, and PPARy. Hypoxia also decreased CYP19 levels, however, further investigation is needed to confirm dihydrotestosterone and flutamide effect on protein expression in trophoblast cells. These data suggest that histone lysine demethylases complex with androgen receptor to regulate androgen responsive genes, including those directing placental vascularization and development. However, further experiments are needed to confirm the necessity of histone lysine demethylases for targeted androgen signaling in trophoblast cells and to determine if androgen directly regulates trophoblast differentiation and invasion. These findings suggest androgen signaling may play a critical role in placental development.
Open Access
Binding of MBNL1 to CUG repeats slows 5'-to-3' RNA decay by XRN2 in a cell culture model of type I myotonic dystrophy
(Colorado State University. Libraries, 2017) Zhang, Junzhen, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Duval, Dawn, committee member; Di Pietro, Santiago, committee member; Yao, Tingting, committee member
Type I myotonic dystrophy (DM1) is a multi-systemic inherited disease caused by expanded CTG repeats within the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. The encoded CUG repeat-containing mRNAs are toxic to the cell and accumulate in nuclear foci, where they sequester cellular RNA-binding proteins such as the splicing factor Muscleblind-1 (MBNL1). This leads to widespread changes in gene expression. Currently, there is no treatment or cure for this disease. Targeting CUG repeat-containing mRNAs for degradation is a promising therapeutic avenue for myotonic dystrophy, but we know little about how and where these mutant mRNAs are naturally decayed. We established an inducible C2C12 mouse myoblast model to study decay of reporter mRNAs containing the DMPK 3' UTR with 0 (CUG0) or ~700 (CUG700) CUG repeats and showed that the CUG700 cell line exhibits characteristic accumulation of repeat-containing mRNA in nuclear foci. We utilized qRT-PCR and northern blotting to assess the pathway and rate of decay of these reporter mRNAs following depletion of mRNA decay factors by RNA interference. We have identified four factors that influence decay of the repeat-containing mRNA – the predominantly nuclear 5' 3' exonuclease XRN2, the nuclear exosome containing RRP6, the RNA-binding protein MBNL1, and the nonsense-mediated decay factor, UPF1. We have discovered that the 5' end of the repeat-containing transcript is primarily degraded in the nucleus by XRN2, while the 3' end is decayed by the nuclear exosome. Interestingly, we have shown for the first time that the ribonucleoprotein complex formed by the CUG repeats and MBNL1 proteins represents a barrier for XRN2-mediated decay. We suggest that this limitation in XRN2-mediated decay and the resulting delay in degradation of the repeats and 3' region may play a role in DM1 pathogenesis. Additionally, our results support previous studies suggesting that UPF1 plays a role in initiating the degradation of mutant DMPK transcripts. This work uncovers a new role for MBNL1 in DM1 and other CUG-repeat expansion diseases and identifies the nuclear enzymes involved in decay of the mutant DMPK mRNA. Our model has numerous applications for further dissecting the pathways and factors involved in removing toxic CUG-repeat mRNAs, as well as in identifying and optimizing therapeutics that enhance their turnover.
Open Access
Characterization of changes in metabolic pathways during dengue virus serotype 2 infection of the Aedes aegypti mosquito vector to identify control points for interrupting virus transmission
(Colorado State University. Libraries, 2018) Chotiwan, Nunya, author; Perera, Rushika, advisor; Blair, Carol, committee member; Foy, Brian, committee member; Huang, Claire, committee member; Di Pietro, Santiago, committee member
Dengue viruses (DENV) are mosquito-borne viruses that cause a wide range of acute symptoms from mild fever to lethal dengue shock syndrome in humans. DENV are transmitted primarily by Aedes aegypti (Ae. aegypti). These mosquitoes are widely distributed throughout tropical and subtropical areas around the world. Increasing globalization, urbanization and global warming are factors that enhance the spread of these vectors placing over 2.5 billion people at risk of contracting these viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate into several tissues in the mosquito vector. During DENV infection of its human and mosquito hosts, a visible rearrangement of lipid membrane architecture and alterations of the metabolic repertoire is induced. These events occur to facilitate efficient viral replication and virus assembly within the cell and to circumvent antiviral responses from the host. Interference with these virus-induced processes can be detrimental to virus replication and can prevent viral transmission. In this dissertation, we present the first insight into the metabolic environment induced during DENV serotype 2 (DENV2) replication in Ae. aegypti. Using untargeted high-resolution liquid chromatography-mass spectrometry, we explored the temporal metabolic perturbations that occur following dengue virus infection of the midgut, the primary site of the virus infection in the mosquito vector. Temporal changes of metabolites across early-, mid- and late-infection time points were identified. A marked increase in the /content of glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels and the accumulation of medium-chain acyl-carnitines suggested a diversion of resources during infection from energy storage to synthetic pathways and energy production. From the observations above, two active pathways, sphingolipid and de novo fatty acid synthesis pathways, were further validated to identify metabolic control hubs. Using inhibitor screening of the sphingolipid pathway, we determined that sphingolipid Δ-4 desaturase (DEGS), the enzyme that converts dihydroceramide to ceramide was important for DENV2 infection in cultured Ae. aegypti cells (Aag2). Long, double-stranded RNA-mediated knockdown of DEGS expression led to the imbalance of ceramide to dihydroceramide ratios and affected DENV2 infection in cell culture. However, the inhibitory effect to DENV2 replication was not observed during DEGS-knockdown in mosquito vectors. De novo fatty acid biosynthesis is the pathway that synthesizes the first lipid molecules, fatty acids, required in synthesizing complex lipid molecules, such as glycerophospholipids, glycerolipids and sphingolipids. As a result, this pathway serves as a bottle neck for the control of lipid metabolism. In this study, we annotated and characterized the expression of seven Ae. aegypti fatty acid synthase (AaFAS) genes in the different stages of mosquito development and upon exposure to different diets. We found that AaFAS1 shares the highest amino acid similarity to human fatty acid synthase (FAS) and is the dominant AaFAS that expressed in female mosquitoes. Knockdown expression of AaFAS1 expression showed a reduction in DENV2 replication in the Aag2 cells and in the midgut of Ae. aegypti mosquitoes during early infection. However, the correlation between viral infection and levels of AaFAS1 expression was difficult to elucidate. The work in this dissertation has highlighted metabolic pathways that are induced by DENV2 infection and the metabolic control points within these pathways that are critical for DENV2 infection in Ae. aegypti. Successful perturbation of metabolic homeostasis can potentially limit virus replication in the vector, presenting a novel avenue to block the transmission of DENV2 from the mosquitoes to humans.
Open Access
Characterization of cyclic nucleotide phosphodiesterases in the transcriptome of the crustacean molting gland
(Colorado State University. Libraries, 2019) Rifai, Nada Mukhtar, author; Mykles, Donald L., advisor; Garrity, Deborah, committee member; Kanatous, Shane, committee member; Di Pietro, Santiago, committee member
Molting in crustaceans is a complex physiological process that has to occur in order for the animal to grow. The old exoskeleton must be discarded and a new one to be formed from the inside out. Molting is coordinated and regulated mainly by two hormones; steroid hormones named ecdysteroids, which are synthesized and secreted from a pair of Y- organs (YOs) that are located in the cephalothorax and a neuropeptide hormone, the molt inhibiting hormone (MIH), which is secreted from the X-organ/sinus gland complex located in the eyestalks. Molting is induced when MIH is decreased in the blood (hemolymph) which in turn stimulates the YOs to produce and secrete ecdysteroids (molting hormones). There are four distinctive physiological states that the YO can be in throughout the molt cycle; the transition of the YO from the "basal" to the "activated" state happens when the animal enters premolt. During mid-premolt, the YO transitions to the "committed" state, in which the YO becomes insensitive to MIH. In this state, the circulating hemolymph contains high levels of ecdysteroids, which increase to a peak before the actual molt (ecdysis) happens. The YO transitions from the committed to the repressed state in late premolt. Finally, the YO returns back to the basal state in the postmolt stage. MIH binds to membrane receptors, activating a signal transduction pathway divided into "triggering" and "summation" phases. A transient increase in cAMP during the triggering phase leads to prolonged cGMP-dependent suppression of ecdysteroidogenesis during the summation phase. This allows for sustained inhibition of the YO between MIH pulses in the intermolt animal. Cyclic nucleotide phosphodiesterases (PDEs) play an important role by controlling cAMP and cGMP levels. PDEs hydrolyze the phosphodiester bond in cAMP and cGMP to AMP and GMP, respectively. Mammals have 21 PDE genes that are categorized into 11 families, designated PDE1 to PDE11. Each PDE family has specific catalytic and biochemical properties and tissue distributions. Eight contigs encoding full-length PDE sequences were identified in the G. lateralis Y-organ transcriptome. Seven contigs encoding four full-length PDE sequences and three contigs encoding partial-length PDE were identified in the Carcinus maenas transcriptome. Multiple sequence alignments showed high sequence identities with orthologs from other species in catalytic (PDEase) and other conserved functional domains. Sequence analysis assigned the Gl-PDE sequences and Cm-PDE sequences to PDE1, PDE2, PDE3, PDE4, PDE5, PDE7, PDE8, PDE9, and PDE11 classes, indicating a high diversity of PDE genes in decapod crustaceans. The reduced sensitivity to MIH by the committed YO is associated with a large increase in PDE activity, which suggests that PDEs modulate the response to neuropeptide during the molt cycle. Non-hydrolyzable analogs of cAMP and cGMP inhibit YO ecdysteroid secretion in-vitro. Moreover, C. maenas YO ecdysteroidogenesis is inhibited by IBMX, a general PDE inhibitor, and Zaprinast, a specific PDE5 inhibitor. Rolipram, a specific PDE4 inhibitor, has no effect. These data suggest that PDE5 activity modulates the effect of MIH on YO ecdysteroidogenesis. RNA-seq data from MLA showed different mRNA levels for the different PDEs; PDE1 and PDE2 showed a similar pattern as they both increased in intermolt (IM) then decreased dramatically in early premolt (EP), mid premolt (MP), late premolt (LP), and post molt (PM). PDE4 increased in IM followed by a slight decrease and increase in EP and MP then a sharp decline in both LP and PM. Both PDE5 and PDE9 were similar in terms they increased in IM followed by a sharp decrease in EP, MP, LP and they differed as PDE5 increased slightly in PM whereas PDE9 remained decreased. PDE7 began with an increase in IM then a decline with a constant expression level in both EP and MP followed by dramatic decline in LP and PM. PDE11 showed a typical pattern consistent with the ecdysteroid expression level as it began with a slight increase in IM followed by an increased in EP and reached a peak in MP then declined in a dramatic way in LP and continued decreasing in PM. Taken together, the data suggest that PDE5 and PDE11 play a role in regulating cyclic nucleotide levels in the YO.
Open Access
Characterization of the selective hydrolysis of branched ubiquitin chains by Uch37 and its activator Rpn13
(Colorado State University. Libraries, 2020) Hazlett, Zachary S., author; Yao, Tingting, advisor; Cohen, Robert, committee member; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Kennan, Alan, committee member
The ubiquitin (Ub) C-terminal hydrolase, Uch37, can be found associated with the 26S proteasome as well as the INO80 chromatin remodeling complex. Bound to the 26S proteasome, it assists in regulating the degradation of Ub modified proteins. The proteasomal subunit Rpn13 binds Uch37, anchors it to the proteasome 19S regulatory particle and enhances the deubiquitinating enzyme's (DUB's) catalytic activity. While the structure of the Uch37/Rpn13 complex bound to a single Ub molecule has been characterized, much still remains unknown regarding the enzyme's substrate specificity, the molecular basis for its substrate specificity, and its function in the regulation of proteasomal degradation. In this thesis we characterize the substrate specificity of Uch37 with and without its proteasomal binding partner Rpn13. By synthesizing poly-Ub chains of various linkage types and topologies and using these Ub chains in in vitro deubiquitination assays, we were able to determine that Uch37/Rpn13 selectively cleaves branched Ub chains. This provides evidence to suggest that Uch37 is the first enzyme with activity specific for branched Ub chains. Branched Ub chains have been identified endogenously and have roles connected to the regulation of nascent misfolded polypeptides, cell cycle control, and the enhancement of proteasomal degradation. The work presented here sets out to characterize the molecular mechanism of branched chain hydrolysis by Uch37 and its binding partner Rpn13, determine the kinetics of this enzymatic reaction, and establish a system for probing the function of "debranching" by Uch37 in proteasomal degradation. The conclusion of our work builds our understanding of the complex system of intracellular signaling by Ub and unveils key elements to the primary system responsible for regulating cellular protein homeostasis.
Open Access
Cofilin-actin rods: quantification and comparison to tau pathology in a human longitudinal aging study and developing probes to measure localization and activity of NADPH oxidase 2, a component of the prion-dependent rod inducing pathway
(Colorado State University. Libraries, 2015) Carlson, Adlei B., author; Bamburg, James R., advisor; Di Pietro, Santiago, committee member; Amberg, Gregory C., committee member
The presence of extracellular amyloid plaques composed mainly of fibrils of the β-amyloid peptide (Aβ) as well as intracellular neurofibrillary tangles composed mainly of hyperphosphorylated tau protein, are used for post-mortem confirmation of the diagnosis of Alzheimer's disease (AD). However, a shift in disease hypothesis has changed over the years. It is now generally accepted that soluble forms of Aβ oligomers and not fibrils, which are deposited in plaques, are most responsible for the synaptic loss and eventual neuronal death that accompanies AD progression. In cultured mammalian neurons, treatment with this more relevant, soluble form of Aβ induces the formation of cofilin-actin rods within neurons. Rods may grow to occlude the neurite and block transport, leading to loss of microtubules and synapses. Tau is a microtubule binding protein whose hyperphosphorylation depends upon its release from microtubules. Thus, rod formation might play a role in the loss of synapses and the development of tau pathology in AD. To determine if cofilin-actin rods might play a role in AD progression, we obtained samples of frontal cortex and the hippocampal formation from nearly identical regions of multiple subjects who were part of a longitudinal study and thus could be grouped as non-cognitively impaired (NCI), early AD (eAD), or mid to late AD. All samples were obtained with a short postmortem interval and the average age of subjects in each group was between 86 and 91 years. We prepared 30 μm sections of cortical and hippocampal tissue, and following immunofluorescence staining for cofilin and phosphorylated tau protein, quantified rod and neuropil thread areas in brain sections from each subject. Rods in the hippocampal formation were most prevalent in the entorhinal cortex, the first brain region to show pathology during development of AD. Comparison of rod and neuropil thread pathology in the frontal cortex revealed a correlation of neuropil thread pathology with disease transition. However, there was no correlation between rod density and disease transition, while the cortical sections revealed a surprisingly high deposition of cofilin rod pathology across all subject cohorts. This may suggest that rods play a different role within brain cortical regions than what was observed in the hippocampus. Additionally, recent work has revealed the implication of a prion-dependent rod inducing pathway dependent on the activation of the reactive oxygen producing NADPH oxidase 2 (NOX). If prion-protein density is responsible for whether a rod forms, can we investigate the NOX intensity and duration of activity in relation to where rods form in a neurite? For future study we sought to develop the sensitive NOX probes, p47-roGFP and NOX-2-redtrack. These probes will give us new tools to analyze the effects that NOX activity and expression have on rod formation. The adenoviral constructs for expression of these two probes have been made and characterized within mammalian cell lines. Evidence presented here provides the basis for the use of these probes to analyze NOX activity as it relates to the generation of rods within neurites.
Open Access
Detection and measurements of free ubiquitin in fixed cells and characterization of OTUB1 contribution to ubiquitin homeostasis
(Colorado State University. Libraries, 2020) Prada Gomez, Luisa Fernanda, author; Cohen, Robert, advisor; Di Pietro, Santiago, committee member; Markus, Steven, committee member; Tamkun, Michael, committee member
Post-translational modifications with Ubiquitin (Ub) have been found to participate in a wide range of cell functions, including protein degradation, endocytosis, regulation of gene expression and cell cycle progression. Therefore, regulation of free Ub levels is essential to ensure that enough Ub is available for conjugation, while excess Ub does not compete in the large number of processes that depend on binding to ubiquitinated proteins or polyUb. Not surprisingly, changes in Ub pool dynamics can affect the cell functions, and perturbations of free Ub levels have been reported to cause neurological and developmental disorders. Although there are techniques to measure Ub pools in vitro, visualization and quantification of free Ub inside individual cells has not been possible. One way to regulate the intracellular concentration of free Ub, is by means of Deubiquitinating enzymes (DUBs), however specific details about the regulatory mechanism are, in large part, unknown. Most studies about DUBs have focused on enzymatic activity and regulation in vitro, with only few reports on the regulation of Ub homeostasis in vivo. The role of OTUB1 in Ub homeostasis has been hypothesized because its catalytic activity is affected by the ratio of [Ub~E2] to [E2] in response to free Ub concentration. Interaction between OTUB1 and a subset of E2s can stimulate OTUB1 isopeptidase activity, whereas interactions with Ub~E2s can inhibit the ubiquitin transfer from the thioester Ub~E2 adduct. This dissertation describes the successful development of a technique to detect and quantify changes in free Ub levels in fixed cells using a high affinity binding protein. The method was used to quantify changes in Ub levels after proteasome and E1 inhibition and to establish the free Ub distribution in hippocampal neurons. It was shown also that OTUB1 activity is not directly involved in the regulation of free Ub levels under stress conditions. However, a new mechanism for regulation of UBE2D expression levels dependent on OTUB1 was identified. This mechanism is independent of proteasomal degradation and could possibly involve translational regulation.
Open Access
Engineering bacteriophage nanocarriers for targeted delivery of protein reagents to prostate cancer cells
(Colorado State University. Libraries, 2014) DePorter, Sandra M., author; McNaughton, Brian, advisor; Kennan, Alan, committee member; Crans, Debbie, committee member; Reynolds, Melissa, committee member; Di Pietro, Santiago, committee member
Proteinaceous reagents, including antibodies and synthetic proteins, have become some of the most effective reagents for targeted treatment and diagnosis of disease. The unique catalytic activity of some proteins and ability to bind disease-relevant receptors that can evade small molecule discovery, make these reagents well suited for use as therapeutic and bioimaging reagents. However, the large size and charge distribution of most proteins greatly inhibits their intracellular delivery to diseased cells, limiting targets to those displayed on the cell surface. In response to this challenge, we have developed a bacteriophage nanocarrier to deliver large payloads of proteinaceous cargo to the interior of prostate cancer cells. This reagent employs two distinct components: a genetically defined prostate cancer cell-selective protein transduction domain, and a biotinylation site on an orthogonal coat protein, which allows for complexation with streptavidin fusion proteins. Collectively, this approach permits targeted intracellular delivery of ~20 exogenous proteins per phage to human prostate cancer cells. This multifunctional technology offers a cell-selective solution to the challenges associated with delivering protein cargo to the interior of diseased cells and may lead to an expansion in the use of protein reagents.
Open Access
Evaluating the effect dynein and related proteins exhibit on the spindle assembly checkpoint and kinetochore
(Colorado State University. Libraries, 2019) Biebighauser, Tyler, author; Markus, Steven, advisor; DeLuca, Jennifer, advisor; Di Pietro, Santiago, committee member; Hoerndli, Fred, committee member
To ensure that cell division is faithfully carried out without causing genetic errors, eukaryotic cells have evolved several conserved checkpoints during mitosis. One such checkpoint, the Spindle Assembly Checkpoint (SAC), blocks the cell from progressing through metaphase until all chromosomes have become bi-oriented by microtubules. Only once this occurs can the cell progress into anaphase to separate the sister chromatids. Errors in this checkpoint have been linked with aneuploidy, which itself is linked with oncogenesis. Naturally there are many layers of regulation within the SAC, most of which are associated with a proteinaceous structure on the sister chromatid – the kinetochore. The molecular motor dynein, and its kinetochore localized co-factors play several roles in this regulation. In one of these roles, dynein strips away kinetochore localized signal proteins upon microtubule bi-orientation, to weaken the strength of the SAC. We initially set out to test whether this process of SAC stripping has further levels of regulation, or if all dynein requires to strip these signal proteins is the presence of a microtubule. We used in-vitro motility assays to investigate whether dynein's motility along microtubules is changed depending on the length of its kinetochore localized cargo adapter, spindly. We purified truncated versions of spindly to test if it undergoes regulation analogous to other dynein cargo adapters. These in-vitro motility assays showed no difference in dynein motility past a certain length required to confer motility. Interestingly, we observed that some of the shorter spindly truncations undergo phase separation both in-vitro in the right conditions and in-vivo when transfected into HeLa cells. We postulate that this phase separation could have implications in a process called fibrous corona expansion, which occurs on a kinetochore that has spent a long time in prometaphase without attaching to a microtubule. In total these studies shed light on the nature of interactions at the kinetochore, and the complexity of regulation as it pertains to dynein mediated kinetochore stripping.
Open Access
Exosomes: a potential novel source of biomarkers for tuberculosis
(Colorado State University. Libraries, 2017) Diaz, Gustavo, author; Dobos, Karen M., advisor; Kruh-Garcia, Nicole, advisor; Belisle, John, committee member; Gonzalez-Juarrero, Mercedes, committee member; Di Pietro, Santiago, committee member
To view the abstract, please see the full text of the document.
Open Access
Flavivirus control of lipid metabolism: implications for virion formation, function and pathogenesis
(Colorado State University. Libraries, 2018) Gullberg, Rebekah, author; Perera, Rushika, advisor; Crick, Dean, committee member; Di Pietro, Santiago, committee member; Geiss, Brian, committee member; Wilusz, Jeff, committee member
Dengue viruses (DENV) are the most aggressive arthropod-born viruses worldwide with no currently available antivirals. There is a clear need to understand host viral interactions that can be exploited for therapeutic options. DENV are members of the Flaviviridae family with a positive sense single-stranded RNA genome surrounded by a virally encoded capsid protein, a host cell derived lipid envelope and an icosahedral shell of virally encoded glycoproteins. Its genome is replicated in virally–induced invaginations in the endoplasmic reticulum of the host cell that consistently develop in a time-dependent manner. These invaginations display a highly curved architecture and seem to increase the membrane contact sites within the ER and its vicinity. Functionally, these membranes condense the replication machinery, provide a scaffold to coordinate replication, and hide the viral double stranded RNA intermediate from the host cellular defenses. It has been shown that fatty acid synthesis is increased during infection to provide substrates for this membrane expansion. To identify further changes to cellular metabolism, we have profiled the metabolome of DENV serotype 2 (DENV2) infected Human Hepatoma cells (Huh7) cells at key time-points in virus replication. We have found time-dependent changes in cellular essential fatty acid metabolism. Furthermore, we have interrogated a library of siRNAs directed at the unsaturated fatty acid biosynthesis pathway to determine key enzymes involved in viral replication. We have identified that stearoyl Co-A desaturase 1 (SCD1), the rate-limiting enzyme responsible for converting stearic to oleic acid, is critical for viral replication, maturation and infectious particle formation. Finally, we have profiled the serum metabolome of acute-phase patients with dengue diseases, chikungunya virus infection, or an unknown febrile illness to identify metabolic changes with potential use as prognostic biomarkers. Hypothesis: Since dengue viruses are enveloped viruses, lipid metabolites in the human host are a critical resource hijacked by these viruses for their replicative advantage. Important metabolites will be altered during infection in a time dependent manner and can be quantified and correlated directly to their role in viral genome replication and infectious particle assembly and release. These metabolic changes could also be identified in human bio-fluids and could function as early biomarkers of disease manifestation.
Open Access
Halogen bonds in biological macromolecules
(Colorado State University. Libraries, 2016) Scholfield, Matthew Robert, author; Ho, P. Shing, advisor; Fisk, John, committee member; Peersen, Olve, committee member; Di Pietro, Santiago, committee member
The purpose of this dissertation is to study how halogen bonds (X-bonds) affect the stability of biological macromolecules and to develop a set of empirical mathematical equations that can provide insight into the anisotropic nature of covalently bound halogens. To achieve this end, we first conducted a detailed analysis of the Protein Data Bank (PDB) to determine the prevalence of X-bonding in biological macromolecules, which allowed us to study the geometrical trends associated with X-bonding. Quantum mechanical (QM) calculations were also applied to determine how the strength of X-bonds interaction could be "tuned." The next chapter used QM calculations to help parameterize an equation that can model the anisotropic size and charge of covalently bound chlorine, bromine and iodine. The energies obtained from this equation were validated on experimentally determined X-bond data by differential scanning calorimetry (DSC) in DNA holiday junctions and were found to nearly duplicate the energies obtained in the solution state experiments. In the final chapter, we engineer X-bonds into the structure of T4 lysozyme to studying structural and thermodynamic effects of X-bonds on protein. X-bonds were introduced into the enzyme via site-specific non-canonical amino acid incorporation and then the structure and stability of the protein were assayed via X-ray crystallography and DSC, respectively. The culmination of this work has elucidated many concepts that need to be considered when trying to engineer new biologically based materials with halogens.
Open Access
High resolution optical analysis of Nav1.6 localization and trafficking
(Colorado State University. Libraries, 2015) Akin, Elizabeth Joy, author; Tamkun, Michael, advisor; Amberg, Gregory, advisor; Di Pietro, Santiago, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member
Voltage-gated sodium (Naᵥ) channels are responsible for the depolarizing phase of the action potential in most nerve cell membranes. As such, these proteins are essential for nearly all functions of the nervous system including thought, movement, sensation, and many other basic physiological processes. Neurons precisely control the number, type, and location of these important ion channels. The density of Naᵥ channels within the axon initial segment (AIS) of neurons can be more than 35-fold greater than that in the somatodendritic region and this localization is vital to action potential initiation. Dysfunction or mislocalization of Naᵥ channels is linked to many diseases including epilepsy, cardiac arrhythmias, and pain disorders. Despite the importance of Naᵥ channels, knowledge of their trafficking and cell-surface dynamics is severely limited. Research in this area has been hampered by the lack of modified Naᵥ constructs suitable for investigations into neuronal Naᵥ cell biology. This dissertation demonstrates the successful creation of modified Naᵥ1.6 cDNAs that retain wild-type function and trafficking following expression in cultured rat hippocampal neurons. The Naᵥ1.6 isoform is emphasized because it 1) is the most abundant Naᵥ channel in the mammalian brain, 2) is involved in setting the action potential threshold, 3) controls repetitive firing in Purkinje neurons and retinal ganglion cells, 4) and can contain mutations causing epilepsy, ataxia, or mental retardation. Using single-molecule microscopy techniques, the trafficking and cell-surface dynamics of Naᵥ1.6 were investigated. In contrast to the current dogma that Naᵥ channels are localized to the AIS of neurons through diffusion trapping and selective endocytosis, the experiments presented here demonstrate that Naᵥ1.6 is directly delivered to the AIS via a vesicular delivery mechanism. The modified Naᵥ1.6 constructs were also used to investigate the distribution and cell-surface dynamics of Naᵥ1.6. Somatic Naᵥ1.6 channels were observed to localize to small membrane regions, or nanoclusters, and this localization is ankyrinG independent. These sites, which could represent sites of localized channel regulation, represent a new Naᵥ localization mechanism. Channels within the nanoclusters appear to be stably bound on the order of minutes to hours, while non-clustered Naᵥ1.6 channels are mobile. Novel single-particle tracking photoactivation localization microscopy (spt-PALM) analysis of Naᵥ1.6-Dendra2 demonstrated that the nanoclusters can be modeled as energy wells and the depth of these interactions increase with neuronal age. The research presented in this dissertation represents the first single-molecule approaches to any Naᵥ channel isoform. The approaches developed during the course of this dissertation research will further our understanding of Naᵥ1.6 cell biology under both normal and pathological conditions.
Open Access
Investigating the regulators of cytoplasmic dynein
(Colorado State University. Libraries, 2019) Dilsaver, Matthew, author; Markus, Steven, advisor; Di Pietro, Santiago, committee member; DeLuca, Jennifer, committee member; Argueso, Juan Lucas, committee member
Organization of the cell is a dynamic and complex process that is often underappreciated. To accomplish this, cells use motor proteins to move different cargo to their destination. Cytoplasmic dynein is one such motor protein that uses filaments called microtubules as tracks. However, there is only one cytoplasmic dynein to accomplish over forty tasks. To achieve this, the cell uses a complex array of cofactors and regulators to specifically control dynein. But the role of each of these cofactors and regulators in poorly understood. To better understand how dynein is regulated we turn to budding yeast that provides a simplified system where dynein only has one known function, this is to position the spindle in the division plane between two dividing cells. Localizing dynein is extremely important. One regulator of dynein is Pac1 which was recently found to also activate dynein motility in vitro. Pac1 works to localize dynein to microtubule plus ends where it can interact with dynactin and Num1. Ndl1 is known to interact with Pac1, knockouts of Ndl1 led to a mild phenotype mimicking a dynein knockout. But how Ndl1 functions is poorly understood. Dynactin is an essential regulator of almost all dynein's tasks in humans and dynein's only role in yeast. Without dynactin, dynein cannot reach Num1 patches at the cell periphery and pull the spindle. In this study we sought to better understand dynactin and Ndl1's role in dynein regulation using in vitro single molecule assays where the activity of dynein can be recorded. Initial attempts to purify dynactin for these assays failed. We then developed a cell lysis assay to study dynactin and other proteins role in dynein regulation. We found in preliminary results that dynactin increased dynein activity. We also attempted to use a protein known as Num1, that is essential to dynein localization and interacts with dynactin, to purify the dynein-dynactin complex. Preliminary results showed that this complex was motile, indicating an intact complex. We also found that Ndl1 can bind motile dynein and increase run length using in vitro assays. We also were able to determine that Pac1 cannot bind dynein and Ndl1 at the same time indicating that there is a release mechanism for Pac1 from Ndl1 to bind dynein. We were able to map Ndl1's binding site to the N terminus of the dynein accessory chain Pac11. Then we tested to see if Ndl1 influence on Pac1-dynein interaction and found that Ndl1 was able to increase Pac1 comigrating with dynein in these assays. This work has opened new strategies for studying the regulators of dynein as well as better determined the interaction between Ndl1, dynein and Pac1. Further work will determine how each of these proteins affect dynein activity.
Open Access
Investigating the roles prion-like domains play in cellular stress responses
(Colorado State University. Libraries, 2018) Shattuck, Jenifer Elizabeth, author; Ross, Eric, advisor; Peersen, Olve, committee member; Di Pietro, Santiago, committee member; Telling, Glenn, committee member
Prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates play an important role in regulating a broad-range of cellular functions. In the budding yeast Saccharomyces cerevisiae, at least 10 proteins have been identified that form self-propagating amyloid-based prions. Most known yeast prion proteins contain a low-complexity, intrinsically-disordered prion-forming domain that is converted into stable, detergent-insoluble aggregates, necessary for prion activity. These prion-forming domains tend to be glutamine/asparagine (Q/N) rich, and relatively lacking in charged and hydrophobic amino acids. To better understand the amino acid sequence features that promote prion activity, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains (PrLD). While from this study we did not identify new yeast prion proteins, we identified several PrLDs with aggregation activity. Therefore, in follow up studies we investigated the role these PrLDs play in other protein assemblies involved in cellular stress responses. First, we investigated how a prion-like protein kinase, Sky1, plays a role in regulating stress granules. Stress granules are cytoplasmic assemblies that form when translation initiation is limiting, including under a variety of stress conditions. Because these cytoplasmic granules are important regulatory machinery for cellular homeostasis, mutations that increase stress granule formation or decrease clearance have been linked to various neurodegenerative diseases. We provided evidence that Sky1 is recruited to stress granules through its aggregation-prone PrLD, and it phosphorylates an RNA-binding protein to efficiently disassemble stress granules. Additionally, we showed when Sky1 is overexpressed it can compensate for defects in other disassembly pathways. These findings contribute to understanding the regulation of stress granules, and provides a possible mechanism to mitigate persistent stress granules in neurodegenerative diseases. Next, we investigated how PrLDs are used to assemble and activate a vacuole-signaling complex. Many cellular processes are regulated primarily through the production of phosphoinositides. Specifically, synthesis and turnover of phosphatidylinositol 3,5 bisphosphate (PtdIns(3,5)P2) is regulated by a vacuole-signaling complex, containing prion-like proteins Fab1, Vac7, and Vac14. Interestingly, during hyperosmotic stress, there is a rapid and dramatic rise in PtdIns(3,5)P2, which leads to vacuole remodeling, critical for cellular survival. We used aggregation-altering mutations to characterize the role of Fab1's PrLD in response to osmotic stress. Overall, these studies provided evidence that Fab1's activation requires its aggregation prone PrLD for recruitment and efficient activation for cellular adaptation to stress. Collectively, the studies described below provide insights into the diverse roles PrLDs play in regulating cellular stress responses. Moreover, these studies have contributed to the field of aggregation-mediated cellular regulation by identifying new proteins involved, new proposed mechanisms, and new insights into the cellular consequences that arise from perturbations in regulation of these processes.
Open Access
Investigation of mechanisms of mitotic recombination in yeast
(Colorado State University. Libraries, 2016) Harcy, Lisa Victoria, author; Argueso, Lucas, advisor; Di Pietro, Santiago, committee member; Liber, Howard, committee member; Suchman, Erica, committee member
At the submicroscopic level within all living cells the workings of a dynamic molecular world attempt to preserve the integrity of DNA - the blueprint of life. This dissertation describes in detail the experimental systems and results from two of our studies conducted in which DNA lesions compromised genomic integrity. The unifying theme of the following chapters revolves around the mechanisms responsible for structural genomic variation, that is, what happens to chromosomes when they break. In the first phase of research, we examined outcomes associated with double-strand breaks (DSBs) at G-quadruplex DNA sequences. Here, we show that G4 DNA motifs, capable of initiating double-strand breaks, result in the formation of chromosomal aberrations. Our results provide structural context and support to a putative mechanism of homology-directed repair revealed by molecular analysis of these complex rearrangements. In the second project, we investigated chromosomal translocations that developed from spontaneously occurring DSBs in diploid yeast to ascertain the means by which the DSBs had been repaired. The objective of this study was to examine the intricate interplay between the non-allelic DSB repair (DSBR) processes: canonical reciprocal homologous recombination (CRHR) vs. break-induced replication (BIR). While numerous assays have previously measured repair pathway efficiency by isolating one of the two pathways from the other, no prior studies to our knowledge, have prospectively examined the contribution of BIR to overall double-strand break repair in diploids, using a non-inducible experimental system where either mechanism can be used freely by the cells. I designed and constructed a new assay system to study chromosomal translocations in the yeast eukaryotic model to investigate the balance between these two DSB repair processes. I characterized genotypic and phenotypic alterations in wild type background and in mutant yeast strains defective for BIR. The data obtained from this work provides an additional perspective to the field of DNA repair biology with broad relevance to DSBR regulation in eukaryotes. It provides further understanding about the role of DNA repair to undesired genetic outcomes, thus, leading the way to the design of new and more effective treatments for diseases in which these molecular actions are the instigators of pathogenesis.
Open Access
Investigation of the sequence features controlling aggregation or degradation of prion-like proteins
(Colorado State University. Libraries, 2017) Cascarina, Sean Micheal, author; Ross, Eric, advisor; Ho, P. Shing, committee member; Di Pietro, Santiago, committee member; Zabel, Mark, committee member
Protein aggregates result from the conversion of soluble proteins to an insoluble form. In some cases, protein aggregates are capable of catalyzing the conversion of their soluble protein counterparts to the insoluble form, resulting in a mode of molecular self-replication. Many of these infectious proteins, or "prions", have been identified and characterized in yeast. This has led to the development of prediction algorithms designed to identify protein domains capable of forming prions. Recently, a number human proteins with aggregation-prone prion-like domains (PrLDs) have been identified, and mutations within PrLDs have been linked to muscular and neurodegenerative disorders. However, the number and diversity of PrLD mutations linked to disease are currently limited. Therefore, the extent to which a broad assortment of PrLD mutations affect intrinsic aggregation propensity, and how well this correlates with aggregation in a cellular context, has not been systematically examined. In Chapter 2, I present evidence suggesting that our prion aggregation prediction algorithm (PAPA) is capable of predicting the effects of a diverse range of mutations on the aggregation propensity of PrLDs in vitro and in yeast. PAPA was also able to predict the effects of many but not all PrLD mutations when the protein was expressed in Drosophila, but with slightly. Therefore, while great strides have been made in predicting intrinsic aggregation propensity, a more complete understanding of the cellular factors that influence aggregation in vivo may lead to further improvement of prion prediction methods. Many intracellular protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Therefore, prions must evade or outcompete these quality control systems in order to form and propagate in a cellular context. However, the sequence features that promote degradation versus aggregation of prion domains and PrLDs have not been systematically defined. In Chapter 3, I present evidence that aggregation propensity and degradation propensity can be uncoupled in multiple ways. First, we find that only a subset of classically aggregation-promoting amino acids elicit a strong degradation response in PrLDs. Second, the amino acids that promoted degradation of the PrLDs did not induce degradation of a glutamine/asparagine (Q/N)-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation, suggesting that protein features surrounding aggregation-prone amino acids can modulate their ultimate effects. Furthermore, degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated role for these residues in yeast prion domains. The protein features that foster susceptibility or resistance to degradation are further explored in Chapter 4. We find that Q/N-rich domains resist degradation in a primary sequence-independent manner, and can even exert a dominant degradation-inhibiting effect when coupled to a degradation-prone PrLD. Furthermore, susceptibility to degradation was a relatively de-centralized feature of the PrLD, requiring a large portion of the domain surrounding degradation-promoting amino acids to permit efficient protein turnover. Collectively, these results provide key insights into the relationship between intrinsically aggregation-prone protein features and the ability to aggregate in the context of intracellular protein quality control factors.
Embargo
miR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila melanogaster
(Colorado State University. Libraries, 2023) Saedi, Hana Ibrahim, author; Tsunoda, Susan, advisor; Hoerndli, Frederic, committee member; Amberg, Gregory, committee member; Di Pietro, Santiago, committee member
miR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. Here, we show that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride and glucose levels and decreased locomotor activity. When challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and significantly prolonged survival. Together, these phenotypes suggest a new role for miR-137 in energy homeostasis. Genetic epistasis experiments show that the starvation resistance of miR-137 mutants involves the insulin signaling pathway, and that loss of miR-137 results in drastically reduced phosphorylation/activation of the single insulin receptor, InR, in Drosophila. We explore the possibility that the protein tyrosine phosphatase61F (PTP61F), ortholog of TC-PTP/PTP1B, known to dephosphorylate InR across species, is a potential in vivo target of miR-137. We show that loss of miR-137 results in upregulation of an endogenously tagged PTP61F protein, and that genetically increasing levels of PTP61F mimics the loss of phosphorylated InR and increased starvation resistance seen in miR-137 mutants. Finally, we show that the enhanced starvation resistance of miR-137 mutants is normalized by activation of the insulin signaling pathway in the nervous system. Our study introduces miR-137 as a new player in the regulation of central insulin signaling and metabolic homeostasis.
Open Access
Molecular basis of [PSI+] yeast prion nucleation
(Colorado State University. Libraries, 2013) Ben Musa, Zobaida A., author; Ross, Eric, advisor; Crans, Debbie, committee member; Zabel, Mark, committee member; Di Pietro, Santiago, committee member
Many fatal diseases arise from the conversion of soluble, functional proteins to insoluble misfolded amyloid aggregates. Amyloid fibers are characterized by filamentous morphology, protease resistance and cross]beta structure. Prions (infectious amyloids) are a specific subset of amyloid fibers, differing from other classes of amyloids by their infectivity. Prions are found in both mammals and yeasts, but there are differences between these two groups. Most yeast prions are characterized by the presence of large numbers of glutamine and asparagine (Q/N) residues, and some other common characteristics have been noted, including the presence of few hydrophobic and charged residues. Although, several attempts have been made with limited success to develop valuable systems to predict prion activity, there is no accurate algorithm that has the ability to predict the prion-forming proteins among the Q/N-rich protein group. In the yeast, it has been shown that amino acid composition, not primary sequence, drives prion activity. Recently, preliminary efforts to define the role of amino acid composition in prion formation have been examined. The fundamental question of this project is how, in yeast Q/N-rich prions, the sequence requirements for nucleation versus propagation differ, and how this information can be used in order to develop a precise prion prediction system. By answering this question we will be able to more accurately identify additional prions in both yeast and other organisms. Our long-term goal in the comprehensive studies of prion formation and propagation mechanisms is to apply this information to mammalian prion diseases. Consequently, we will be able to identify targets for therapeutic intervention to avoid, slow-down, or reverse the development of related diseases. The study determined that the amino acids required for prion formation differ from those required for prion propagation. Identifying the sequence feature for both activities is the first step towards mechanistic studies to examine how these sequences perform their function.