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- Title
- In Vitro Characterization of Unmodified and Pyroglutamylated Alzheimer's Amyloid beta peptide.
- Creator
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Matos, Jason, Tatulian, Suren, Teter, Kenneth, Davidson, Victor, University of Central Florida
- Abstract / Description
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Plaques of amyloid ? peptide (A?) are a hallmark trait of Alzheimer's disease (AD). However, the precise role of A? aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of A? significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated A? (pE-A?) in AD brains has been shown to reach up to 50% of total A?. Modified pE-A? co-aggregates with A? by a seeding mechanism and forms...
Show morePlaques of amyloid ? peptide (A?) are a hallmark trait of Alzheimer's disease (AD). However, the precise role of A? aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of A? significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated A? (pE-A?) in AD brains has been shown to reach up to 50% of total A?. Modified pE-A? co-aggregates with A? by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length A?1-42, its pyroglutamylated form A?pE3-42, their 9:1 (A?1-42/A?pE3-42) and 1:1 molar combinations. Transmission electron microscopy was used to directly visualize the fibrils of the samples in a buffer mimicking physiological environment. Atomic force microscopy measurements were done to determine rate of second nucleation events in fibrils. Thioflavin-T fluorescence indicated that low ionic strength suppressed the aggregation of A?pE3-42 but promoted that of A?1-42, suggesting different paths of fibrillogenesis of unmodified A? and pE-A?. Interestingly, A?pE3-42 at only 10% significantly facilitated the fibrillization of A?1-42 at near physiological ionic strength but had little effect at low salt. Circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used to characterize the structural transitions during fibrillogenesis. In aqueous buffer, both unmodified A? and pE-A? peptides adopted parallel intermolecular ?-structure. Interestingly, A?pE3-42 contained lower ?-sheet content than 13C-A?1-42, while retaining significantly larger fractions of ?-helical and turn structures. Structural details of A? and pE-A? combinations were unveiled by isotope-edited FTIR spectroscopy, using 13C-labeled A?1-42 and unlabeled A?pE3-42. When exposed to environmental humidity, A?pE3-42 not only maintained an increased fraction of ?-helix but also was able to reverse 13C-A?1-42 ?-sheet structure. These data provide a novel structural mechanism for pE-A? hypertoxicity; pE-A? undergoes fasternucleation due to its increased hydrophobicity, thus promoting formation of smaller, hypertoxic oligomers of partial ?-helical structure.
Show less - Date Issued
- 2014
- Identifier
- CFE0005378, ucf:50465
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005378
- Title
- Antimicrobial Peptide Resistance and Immunomodulation by HIV-1 gp41.
- Creator
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Wood, Matthew, Cole, Alexander, Chai, Karl, Teter, Kenneth, Parkinson, Christopher, University of Central Florida
- Abstract / Description
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Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity....
Show moreFusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the ?-defensin analog RC-101 is being developed as a microbicide to prevent sexual transmission of HIV-1. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development.Partial drug resistance due to genetic variability within HIV-1 presents a major hurdle in microbicide development. Drug-resistance mutations, whether naturally occurring or resulting from selection during treatment, often apply to many drugs in a particular class. Combining different drug classes into a single microbicide should provide greater protection against the growing variability observed in HIV. Our work has identified the beneficial effects of combining the fusion inhibitor RC-101 and the RT inhibitor CSIC to prevent transmission of clinically isolated and drug-resistant HIV-1.Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. While this chemotactic activity has been characterized, it is not known how these peptides could be produced under biological conditions. Our findings demonstrate that the epithelial serine protease matriptase efficiently cleaves the gp41 HR1 region at conserved residues into a chemotactic peptide.Here, we present evidence that advances our understanding of resistance to peptide entry inhibitors, reveals a potential benefit to combining specific drugs in an antiviral microbicide, and identifies a pathway by which HIV-1 may generate peptides to exploit host immunity. This work thereby facilitates improved methods in countering drug resistance and the development of new antiviral approaches to prevent HIV-1 transmission. Additionally, we have revealed basic mechanistic evidence that shed light on our current understanding of HIV-1 infection. Specifically, our focus on gp41 provides much needed insight into its role in membrane fusion, drug susceptibility, and modification of host responses.
Show less - Date Issued
- 2014
- Identifier
- CFE0005560, ucf:50286
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005560
- Title
- A chemical and genetic approach to study the polyamine transport system in Drosophila.
- Creator
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Wang, Minpei, Vonkalm, Laurence, Phanstiel, Otto, Teter, Kenneth, Ballantyne, John, University of Central Florida
- Abstract / Description
-
Polyamines are small cationic molecules that play important roles in most vital cellular processes including cell growth and proliferation, regulation of chromatin structure, translation and programmed cell death. Cellular polyamine pools are maintained by a balance between biosynthesis and transport (export and import). Increased polyamine biosynthesis activity and an active transport system are characteristics of many cancer cell lines, and polyamine depletion has been shown to be a viable...
Show morePolyamines are small cationic molecules that play important roles in most vital cellular processes including cell growth and proliferation, regulation of chromatin structure, translation and programmed cell death. Cellular polyamine pools are maintained by a balance between biosynthesis and transport (export and import). Increased polyamine biosynthesis activity and an active transport system are characteristics of many cancer cell lines, and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by ?-difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase. However, malignant cells often circumvent DFMO therapy by up-regulating polyamine import; therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and polyamine transport inhibitors provide the basis for a combination therapy leading to effective intracellular polyamine depletion. Using a Drosophila leg imaginal disc model for polyamine transport, I studied three candidate transport inhibitors (Ant444, Trimer44 and Triamide44) for their ability to inhibit transport in the Drosophila model. Ant444 and Trimer44 effectively inhibited the uptake of the toxic polyamine analog Ant44 that gains entry to cells via the polyamine transport system. Ant444 and Trimer44 were also able to inhibit the import of exogenous polyamines into DFMO-treated imaginal discs. Triamide44 was an ineffective inhibitor, however a structurally redesigned compound, Triamide444, showed a 50-fold increase in transport inhibition and was comparable to Ant444 and Trimer44. Ant444 and Trimer44 showed differences in their relative abilities to block import of specific polyamines, and I therefore asked if a cocktail of these inhibitors would be more effective than either alone. My data show that a cocktail of polyamine transport inhibitors is more effective than single inhibitors when used in combination with DFMO, and suggests the existence of multiple polyamine transport systems. To further the development of effective transport inhibitors it is important to identify components of the transport system. The mechanism of polyamine transport in multicellular organisms including mammals is still unknown. Our laboratory has developed a simple assay to detect components of the transport system using RNAi knockdown and over-expression of candidate genes. However, the assay requires that animals live until the pupal stage of development. Pleiotropic effects of individual gene products following over-expression or knockdown may result in early developmental lethality for reasons unrelated to polyamine transport. Our assay is based on the GAL4/UAS system and involves the use of enhancers driving GAL4 expression (GAL4 driver). GAL4 in turn determines the expression level of UAS-candidate gene constructs (UAS responder). I reasoned that in some cases it might be possible to bypass early lethality by judicious choice of drivers that reduce responder expression, thus permitting survival to the pupal phase. To this end, I used five imaginal disc drivers (30A, 71B, 32B, 69B, and T80) as well as a ubiquitously expressed control driver to over-express and knockdown EGFR and components of the Rho signaling pathway. The relative strength of each driver was ranked, and I was able to demonstrate in principle that animals could survive to later stages of development in a manner that correlated with the relative strength of the driver. The approach I developed is broadly applicable to other studies of Drosophila development.To identify new components of the polyamine transport system I studied the role of proteoglycans in this process. The proteoglycan glypican-1 has been previously implicated in mammalian polyamine transport. In particular, the heparin sulfate side chains of glypican-1 appear to play an important role. In order to extend our knowledge of the role of proteoglycans in polyamine transport, I examined the role of the core proteoglycans perlecan and syndecan as well as genes encoding enzymes in the heparin sulfate and chondroitin sulfate biosynthetic pathways. I was able to confirm a role for glypican-1 in polyamine transport in imaginal discs but not in whole animals. This may indicate that glypican-1 is not required for polyamine uptake through the gut. Studies of genes encoding perlecan, syndecan and enzymes in the heparin sulfate and chondroitin sulfate biosynthetic pathways did not reveal a role for these genes in polyamine transport. These studies were conducted in whole animals and my data may reflect tissue-specific differences between the imaginal disc and gut transport systems where transport in imaginal discs is proteoglycan dependent and transport in the gut is not.
Show less - Date Issued
- 2017
- Identifier
- CFE0007297, ucf:52162
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007297
- Title
- Split Aptameric Turn-On Fluorescence Sensor for Detection of Sequence Specific Nucleic Acid.
- Creator
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Kikuchi, Nanami, Kolpashchikov, Dmitry, Zhai, Lei, Chumbimuni Torres, Karin, Chen, Gang, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Nucleic acid amplification tests (NAATs) enable sensitive detection of low density infections that microscopy and rapid diagnostic test are not capable of detecting. They enable quantitative and qualitative nucleic acid detection, genotype analysis, and single nucleotide polymorphism (SNP) detection. Current state of the art molecular probes used with NAATs includes molecular beacon (MB), Taqman and its variations. This work presents novel molecular probe designed from Spinach and Dapoxyl...
Show moreNucleic acid amplification tests (NAATs) enable sensitive detection of low density infections that microscopy and rapid diagnostic test are not capable of detecting. They enable quantitative and qualitative nucleic acid detection, genotype analysis, and single nucleotide polymorphism (SNP) detection. Current state of the art molecular probes used with NAATs includes molecular beacon (MB), Taqman and its variations. This work presents novel molecular probe designed from Spinach and Dapoxyl aptamers. The aptamers are split into two parts (split aptamer), allowing greater sensitivity and selectivity towards fully complementary nucleic acid analyte. The major advantage of split aptamer probe over state-of-the-art fluorescent probes is its high selectivity: in the presence of a single base mismatched analyte, it produces only background fluorescence, even at room temperature. SSA is a promising tool for label-free analysis of nucleic acids at ambient temperatures.Split spinach aptamer (SSA) probes and split dapoxyl aptamer (SDA) for fluorescent analysis of nucleic acids were designed and tested. In both split aptamer design, two RNA or RNA/DNA or DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for fluorescent dye, which was accompanied by up to 270-fold and 69-fold increase in fluorescence. SSAr consisted entirely of ribonucleotides which potentially be expressed in live cells and used for imaging of specific mRNAs. For in vitro RNA/DNA analysis, SDA consisting of entirely DNA are preferable due to greater chemical stability, lower synthetic cost and reduced ability to form intramolecular structures. Additionally, we designed two DNA strands that function as an adapter for SSA and demonstrated how a single universal spinach aptamer (USSA) probe can be used to detect multiple (potentially any) nucleic acid sequences. USSA can be used for cost-efficient and highly selective analysis of even folded DNA and RNA analytes, as well as for the readout of outputs of DNA logic circuits.
Show less - Date Issued
- 2018
- Identifier
- CFE0007031, ucf:51976
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007031
- Title
- Manipulation of host signal transduction pathways and cytoskeleton functions by invasive bacterium Listeria monocytogenes and Chlamydia trachomatis.
- Creator
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Jiwani, Shahanawaz, Jewett, Travis, Zervos, Antonis, Khaled, Annette, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Infectious disease remains one of the leading causes of morbidity and mortality worldwide. Many bacteria that cause disease have the capacity to enter into eukaryotic cells such as epithelial cells and tissue macrophages. Gaining access into the intracellular environment is one of the most critical steps in their survival and/or in pathogenesis. The entry mechanisms employed by these organisms vary considerably, but most mechanisms involve sabotaging and manipulating host cell functions....
Show moreInfectious disease remains one of the leading causes of morbidity and mortality worldwide. Many bacteria that cause disease have the capacity to enter into eukaryotic cells such as epithelial cells and tissue macrophages. Gaining access into the intracellular environment is one of the most critical steps in their survival and/or in pathogenesis. The entry mechanisms employed by these organisms vary considerably, but most mechanisms involve sabotaging and manipulating host cell functions. Invasion of epithelial cells involves triggering host signal transduction mechanisms to induce cytoskeleton rearrangement, thereby facilitating bacterial uptake. My work focuses on understanding the molecular mechanisms employed by bacterial pathogen Listeria monocytogenes and Chlamydia trachomatis to gain access into the host cells in order to cause the disease.In first part of my thesis I investigated the mechanism of Listeria monocytogenes entry. Listeria, a facultative intracellular organism, is responsible for causing meningitis, septicemia, gastroenteritis and abortions. Critical for Listeria virulence is its ability to get internalized, replicates and spread into adjacent host cells. One of the pathways of Listeria internalization into mammalian cells is promoted by binding of its surface protein Internalin B (InlB) to host receptor MET. Studies done in the past demonstrated a critical role of host type IA Phosphoinositide (PI) 3-kinase in controlling cytoskeleton rearrangement and entry of Listeria downstream of MET. An important unresolved question was how activation of PI3K results in cytoskeleton rearrangements that promote Listeria entry. In this work, we identified 9 host signaling molecules, that includes Rab 5c, SWAP 70, GIT1, PDK1, mTor, ARAP2, ARNO, DAPP1 (&) PKC-?, acting downstream of type IA Phosphoinositide (PI) 3-kinase to regulate changes in host cytoskeleton to cause Listeria entry.Second part of my thesis involved studying the functions of chlamydial effector protein Tarp in its invasion. Infection caused by Chlamydia Trachomatis is the most common sexually transmitted disease resulting in uro-genital diseases, LGV, ectopic pregnancy and infertility. It is also responsible for causing trachoma, the leading cause of preventable blindness in third world countries. Being an obligate intracellular pathogen, gaining access into intracellular environment is the most critical step in lifecycle and pathogenesis of Chlamydia. Previous studies demonstrate the role of both chlamydial and host actin nucleators, Tarp and Arp2/3 complex respectively, in mediating Chlamydial entry into non-phagocytic cells. But the molecular details of these processes were not well understood. In this study, we demonstrate novel function of Tarp protein to form actin bundles by its ability to bind filamentous actin through newly identified FAB domains. And we also provide bio-chemical evidence that Tarp and Arp2/3 complex works in conjunction to cause changes in host cytoskeleton that effectively culminate into bacterial uptake by host cells.Overall, this research was a significant step in enhancing our understanding, at a molecular level, to pathogenesis of infections caused by Listeria monocytogenes and Chlamydia trachomatis.
Show less - Date Issued
- 2012
- Identifier
- CFE0004555, ucf:49225
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004555
- Title
- The Role of Mitochondrial Omi/HtrA2 Protease in Protein Quality Control and Mitophagy.
- Creator
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Ambivero, Camilla, Zervos, Antonis, Teter, Kenneth, Siddiqi, Shadab, Self, William, University of Central Florida
- Abstract / Description
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Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors...
Show moreOmi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a Lys-63 deubiquitinating (DUB) complex. In addition, we found the mechanism by which Omi/HtrA2 protease participates in mitophagy by directly regulating the protein level of Mulan E3 ubiquitin ligase, especially during mitochondrial stress.Abro1 is a scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex). In addition, Abro1 is involved in a cytoprotective pathway and is regulated by Omi/HtrA2. Three specific interactors of Abro1 protein were identified, ATF4, ATF5 and JunD, all members of the activating protein 1 (AP-1) family. We focused our studies on ATF4 since, like Abro1, it is ubiquitously expressed and is important in cell cycle regulation and survival. Abro1's interaction with ATF4 was specific and occurred only when cells were stressed. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the cell nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex.Furthermore, we have recently identified the mitochondrial Mulan E3 ubiquitin ligase as a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death, and autophagy/mitophagy. To investigate Mulan's function and its control by Omi/HtrA2, E2 conjugating enzymes that form a complex with Mulan E3 ligase were identified. Four specific interacting E2s were isolated, namely Ube2E2, Ube2E3, Ube2G2, and Ube2L3. To identify substrates for each unique Mulan-E2 complex, fusion baits were used in a modified yeast two-hybrid screen. Our results suggest that Mulan participates in various pathways, depending on the nature of its E2 conjugating enzyme partner. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP (GABAA receptor-associated protein), a member of the Atg8 family. We characterized this interaction both in vitro and in vivo and its potential role in mitophagy. Our studies defined a new pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria.
Show less - Date Issued
- 2013
- Identifier
- CFE0004805, ucf:49752
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004805
- Title
- The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.
- Creator
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Bader, Carly, Teter, Kenneth, Zervos, Antonis, Jewett, Travis, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much...
Show moreCholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals.The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.
Show less - Date Issued
- 2013
- Identifier
- CFE0004810, ucf:49759
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004810
- Title
- The role of a highly conserved eubacterial ribosomal protein in translation quality control.
- Creator
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Naganathan, Anusha, Moore, Sean, Cole, Alexander, Teter, Kenneth, Roy, Herve, Koculi, Eda, University of Central Florida
- Abstract / Description
-
The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of...
Show moreThe process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions.Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.
Show less - Date Issued
- 2015
- Identifier
- CFE0005674, ucf:50169
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005674
