EFEMP1 suppresses malignant glioma growth and exerts its action within the tumor extracellular compartment

<p>Abstract</p> <p>Purpose</p> <p>There are conflicting reports regarding the function of EFEMP1 in different cancer types. In this study, we sought to evaluate the role of EFEMP1 in malignant glioma biology.</p> <p>Experimental Design</p> <p>Real-time qRT-PCR was used to quantify <it>EFEMP1 </it>expression in 95 glioblastoma multiforme (GBM). Human high-grade glioma cell lines and primary cultures were engineered to express ectopic EFEMP1, a small hairpin RNA of EFEMP1, or treated with exogenous recombinant EFEMP1 protein. Following treatment, growth was assayed both <it>in vitro </it>and <it>in vivo </it>(subcutaneous (s.c.) and intracranial (i.c.) xenograft model systems).</p> <p>Results</p> <p>Cox regression revealed that EFEMP1 is a favorable prognostic marker for patients with GBM. Over-expression of EFEMP1 eliminated tumor development and suppressed angiogenesis, cell proliferation, and VEGFA expression, while the converse was true with knock-down of endogenous EFEMP1 expression. The EFEMP1 suppression of tumor onset time was nearly restored by ectopic VEGFA expression; however, overall tumor growth rate remained suppressed. This suggested that inhibition of angiogenesis was only partly responsible for EFEMP1's impact on glioma development. In glioma cells that were treated by exogenous EFEMP1 protein or over-expressed endogenous EFEMP1, the EGFR level was reduced and AKT signaling activity attenuated. Mixing of EFEMP1 protein with cells prior to s.c. and i.c. implantations or injection of the protein around the established s.c. xenografts, both significantly suppressed tumorigenicity.</p> <p>Conclusions</p> <p>Overall, our data reveals that EEFEMP1 suppresses glioma growth <it>in vivo</it>, both by modulating the tumor extracellular microenvironment and by altering critical intracellular oncogenic signaling pathways.</p

A study of colloidal asphaltene in petroleum reservoirs

Imperial Users onl

A study of colloidal asphaltene in petroleum reservoirs

SIGLEAvailable from British Library Document Supply Centre-DSC:98/21491 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Exploring Candida biology through integration of proteomic and genetic approaches

Candida albicans is a common opportunistic fungal pathogens of humans. As such, it faces challenges unique to host-dwelling organisms and has evolved mechanisms that allow it to thrive in the constantly changing environment of the human host. In this dissertation we sought to address two such mechanisms of C. albicans biology.C. albicans can undergo phenotypic switching between two heritable states: white and opaque. This phenotypic plasticity facilitates its colonization in distinct host niches. The master regulator WOR1 is exclusively expressed in opaque phase cells. Positive feedback regulation by Wor1 on the WOR1 promoter is essential for opaque formation, however the underlying mechanism of how Wor1 functions is not clear. In Chapter 3, we use tandem affinity purification coupled with mass spectrometry to identify Wor1-interacting proteins. Tup1 and its associated complex proteins are found as the major factors associated with Wor1. Tup1 occupies the same regions of the WOR1 promoter as Wor1 preferentially in opaque cells. Loss of Tup1 is sufficient to induce the opaque phase, even in the absence of Wor1. This is the first such report of a bypass of Wor1 in opaque formation. These genetic analyses suggest that Tup1 is a key repressor of the opaque state and Wor1 functions to alleviate Tup1 repression at the WOR1 promoter. Opaque cells convert to white en masse at 37°C. However, we find this conversion occurs only in the presence of glycolytic carbon sources, and the opaque state is stabilized when cells are cultured on non-glycolytic carbon sources, even in an MTLa/α background. We further show that temperature and carbon source affect opaque stability by altering the levels of Wor1 and Tup1 at the WOR1 promoter. We propose that Wor1 and Tup1 form the core regulatory circuit controlling the opaque transcriptional program. This model provides molecular insights on how C. albicans adapts to different host signals to undergo phenotypic switching for colonization in distinct host niches.Iron is an essential molecule involved in a myriad of biological processes. Despite its essential role as a cofactor, excess iron can become toxic through the generation of reactive oxygen species. As a fungal pathogen of humans, C. albicans is subject to a wide range of iron levels encountered in the human host. Consequently, iron homeostasis is essential for survival and is tightly controlled by a regulatory circuit. Glutaredoxins are a conserved family of proteins involved in maintaining cellular redox homeostasis as well as the biogenesis of iron-sulfur clusters, cofactors linked to diverse biological processes including metabolism, DNA maintenance, transcriptional regulation, and protein translation. In Chapter 4, using a combination of functional genetics, molecular biology, and cross-linked tandem affinity purification coupled with mass spectrometry, we assess the function of the conserved monothiol glutaredoxin Grx3. We examined the function of four identified C. albicans glutaredoxins in response to oxidative and iron stresses and found a grx3 mutant to be sensitive to iron level. The grx3 mutant was defective in the expression of the iron-regulatory circuit genes Sfu1, Sef1, and Hap43 in response to iron status. We determined that Grx3 interacts with Sfu1 and regulates its occupancy at SEF1 promoter. Grx3 was also found to contribute to Sef1 nuclear localization and Hap43 activity. Therefore, Grx3 directly controls the activity of the iron homeostasis regulatory circuit. Identification of Grx3 interacting proteins by mass spectrometry uncovered proteins enriched for several functional categories, including those involved in various metabolic and biosynthetic pathways such as redox homeostasis, amino acid and nucleotide metabolism, protein translation, tRNA aminoacylation DNA maintenance and repair, iron-sulfur biogenesis, and iron homeostasis. We validate some of these findings and show that the grx3 mutant is hypersensitive to oxidative, nitrosative and genotoxic stresses and shows decreased translational efficiency compared to wild-type. Finally, we show the grx3 mutant displays decreased virulence in a disseminated infection model. Therefore, we propose C. albicans Grx3 is a global iron sensor critical for both the regulation of the iron homeostasis circuit as well as the functions of iron-sulfur cluster containing proteins involved in a wide array of diverse biological processes

Wor1 establishes opaque cell fate through inhibition of the general co-repressor Tup1 in Candida albicans.

The pathogenic fungus Candida albicans can undergo phenotypic switching between two heritable states: white and opaque. This phenotypic plasticity facilitates its colonization in distinct host niches. The master regulator WOR1 is exclusively expressed in opaque phase cells. Positive feedback regulation by Wor1 on the WOR1 promoter is essential for opaque formation, however the underlying mechanism of how Wor1 functions is not clear. Here, we use tandem affinity purification coupled with mass spectrometry to identify Wor1-interacting proteins. Tup1 and its associated complex proteins are found as the major factors associated with Wor1. Tup1 occupies the same regions of the WOR1 promoter as Wor1 preferentially in opaque cells. Loss of Tup1 is sufficient to induce the opaque phase, even in the absence of Wor1. This is the first such report of a bypass of Wor1 in opaque formation. These genetic analyses suggest that Tup1 is a key repressor of the opaque state, and Wor1 functions via alleviating Tup1 repression at the WOR1 promoter. Opaque cells convert to white en masse at 37°C. We show that this conversion occurs only in the presence of glycolytic carbon sources. The opaque state is stabilized when cells are cultured on non-glycolytic carbon sources, even in a MTLa/α background. We further show that temperature and carbon source affect opaque stability by altering the levels of Wor1 and Tup1 at the WOR1 promoter. We propose that Wor1 and Tup1 form the core regulatory circuit controlling the opaque transcriptional program. This model provides molecular insights on how C. albicans adapts to different host signals to undergo phenotypic switching for colonization in distinct host niches

Tup1 depletion stabilizes the opaque state even at 37°C.

<p>(<b>A)</b> Time course of Wor1 and Tup1 protein levels in opaque cells after shift from room temperature to 37°C. Overnight cultures of opaque cells of a strain carrying both Wor1-FLAG and Tup1-HA (HLY4541) were inoculated, grown to mid-log phase, then shifted to 37°C and grown for the indicated times. Protein level was assessed by Western blot as described. <b>(B)</b> ChIP of Wor1-FLAG and Tup1-HA at the <i>WOR1</i> promoter in opaque cells shifted to room temperature or 37°C. Overnight cultures of opaque cells of a strain carrying both Wor1-FLAG and Tup1-HA (HLY4541) and an untagged control strain (JYC1) were diluted in SCD and grown to log phase at room temperature. Cultures were divided and incubated at either room temperature or 37°C for one hour, formaldehyde cross-linked, and harvested for ChIP. Enrichment is presented as a ratio of the -4kb region of the <i>WOR1</i> promoter IP (bound/input) over an <i>ADE2</i> control region IP (bound/input) of the tagged strain, further normalized to the control strain. Values are the average of three independent ChIP experiments with error bars representing the s.d. <b>(C)</b> Tup1 depletion in opaque cells at room temperature and 37°C. Opaque <i>pMET3-TUP1</i> cells were grown in SCD with or without methionine at either room temperature or 37°C for 24hr. Expression levels of the indicated genes were measured by qPCR and normalized to <i>ACT1</i>. Average expression level of three independent qPCR experiments are plotted with error bars representing the s.d. Samples were also taken at the indicated times, washed three times with H₂O, and plated onto SCD Met- plates to assess phase switching.</p

Tup1 depletion bypasses the requirement for Wor1 in the expression of <i>WOR1</i> and key opaque regulators.

<p><b>(A)</b> Expression levels in the conditional Tup1 mutant and <i>wor1 pMET3-tup1</i> conditional double mutant haploid strains. <i>pMET3-TUP1</i> (HLY4533) and <i>pMET3-TUP1 wor1</i> (HLY4539) were grown at room temperature for 48hr in the presence or absence of methionine. <b>(B)</b> Expression levels in diploid <i>wor1</i> and <i>wor1 tup1</i> mutant strains. Overnight cultures of <i>MTL</i><b><i>a</i></b><i>/</i><b><i>a</i></b> <i>wor1</i> (HLY3570), <i>wor1 tup1</i> (HLY4540), and a control strain (JYC1) were inoculated into fresh SCD and grown to log phase. Expression levels of the indicated genes in <b>(A)</b> and <b>(B)</b> were measured by qPCR and normalized to <i>ACT1</i>. <i>WOR1</i> expression was measured by qPCR using primers specific to either the <i>WOR1</i> 5’ UTR or the <i>WOR1</i> coding region (CDS). Average expression level of three independent qPCR experiments was plotted with error bars representing the s.d.<b>(C)</b> Genetic model of Wor1-Tup1 regulation of <i>WOR1</i> expression.</p

Non-glycolytic carbon sources alter Tup1 occupancy at the <i>WOR1</i> promoter and stabilize the opaque phase at 37°C in <i>MTL</i>a/a and a/α cells.

<p><b>(A)</b> Opaque stability of <i>MTL</i><b>a</b>/<b>a</b> cells cultured in various carbon sources at 37°C for 24hr. Overnight cultures of <i>MTL</i><b>a</b>/<b>a</b> WT opaque cells (HLY3555) grown in SCD were washed three times with H₂O and inoculated into fresh SC medium containing the indicated carbon sources. Cultures were grown at room temperature for 3hr then transferred to 37°C for 24hr. Samples were plated onto SCD plates and grown for 5–7 days to assess phase switching. <b>(B)</b> ChIP of Wor1 and Tup1 in opaque cells at room temperature and 37°C in different carbon sources. Opaque cells carrying both Wor1-FLAG and Tup1-HA (HLY4541) and an untagged strain (JYC1) were grown in SC medium containing the indicated carbon source overnight at room temperature. Cultures were diluted and grown to log phase, then grown at either room temperature or 37°C for 1hr for ChIP. Enrichment is presented as a ratio of the -4kb region of the <i>WOR1</i> promoter IP (bound/input) over an <i>ADE2</i> control region IP (bound/input) of the tagged strain, further normalized to the control strain. Values are the average of three independent ChIP experiments with error bars representing the s.d. (<b>C)</b> Opaque stability of <i>MTL</i><b><i>a</i></b><i>/α</i> cells cultured in liquid media at 37°C for 24hr. Overnight cultures of opaque <i>MTL</i><b><i>a</i></b><i>/α</i> cells carrying <i>pMAL2-WOR1</i> (HLY4543) from SCM were washed three times with H₂O and inoculated into YNB medium containing the indicated carbon sources. Cultures were grown for 3hr at room temperature then shifted to 37°C. Cells were collected after 24hr and gene expression levels were analyzed by qPCR and normalized to <i>ACT1</i>. Average expression level of three independent qPCR experiments are plotted with error bars representing the s.d. <b>(D)</b> Opaque stability of <i>MTL</i><b><i>a</i></b><i>/α</i> cells on solid media. Overnight cultures of opaque <i>MTL</i><b><i>a</i></b><i>/α</i> cells carrying <i>pMAL2-WOR1</i> (HLY4543) grown in SCM were washed three times with H₂O then plated onto YNB plates containing 2% of the indicated carbon source. Plates were incubated at room temperature or 37°C for 5–7 days and scored for percent opaque. Both whole and sectored opaque colonies were counted as opaque.</p