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Browsing by Author "Cohen, Bob, committee member"

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    Bioengineering of cloneable inorganic nanoparticles
    (Colorado State University. Libraries, 2023) Hendricks, Alexander Ryo, author; Ackerson, Christopher J., advisor; Chung, Jean, committee member; McNally, Andy, committee member; Cohen, Bob, committee member
    When a defined protein/peptide (or combinations thereof) control and define the synthesis of an inorganic nanoparticle, the result is a cloneable NanoParticle (cNP). This is because the protein sequence/structure/function is encoded in DNA, and therefore the physicochemical properties of the nanoparticle are also encoded in DNA. Thus the cloneable nanoparticle paradigm can be considered as an extension of the central dogma of molecular biology (e.g. DNA -> mRNA -> Protein -> cNP); modifications to the DNA encoding a cNP can modify the resulting properties of the cNP. The DNA encoding a cNP can be recombinantly transferred into any organism. Ideally, this enables recombinant production of cNPs with the same defined physiochemical properties. Such cNPs are of primary interest for applications in biological imaging as clonable contrast agents. The advancement of cNPs for broader and more rigorous applications in imaging (and elsewhere) requires further development through multidisciplinary approaches. Described in This Thesis is a bioengineering approach to improve the cNP platform through development of the enzymes responsible for nanoparticle formation.In the first chapter, background and significance is given to provide rationale behind the cNP platform. Among all the modalities of biological imaging, there is no 'one-size-fits-all' solution. Biological fluorescence microscopy (FM) and electron microscopy (EM) are the preferred methods of choice when imaging at cellular levels. Although the relatively recent advent of fluorescent proteins and super-resolution microscopy have ushered major scientific breakthroughs, FM is resolution-limited: only the cellular components which are labeled by fluorophores can be resolved – everything else in a cell (~99% of components) is imaged with low resolution owing to the diffraction limit of light. Biological EM comparatively can image widefield cells at atomic-level resolution yet lacks an analogous toolset to fluorescent proteins. cNPs are proposed as a multimodal, uniform, and precise means of clonable contrast for biological EM (and other modalities) analogous to fluorescent proteins. In the second chapter, a tellurium reductase is isolated and characterized from screened environmental bacterial cultures collected throughout the Colorado Mineral Belt. A strain of Rhodococcus erythropolis PR4 was found to be highly resistant to a broad range of metal(loid) species at toxic concentrations – notably 4.5 mM TeO32− determined by broth microdilution. Through screening of cell lysate in the presence of metal(loid) substrates, a mycothione reductase was characterized as a Te-specialized enzyme which reduces Te preferentially over Se. This is a surprising finding on the basis of reduction potentials for the two substrates. The standard reduction of potential for the reaction TeO32− + 3 H2O + 4e− ← → Te + 6 OH− is −0.57 V vs Hydrogen. The corresponding reduction of SeO32− is −0.366 V. Thus, SeO32− is the preferred substrate for reduction in the absence of a mechanism for substrate selectivity. We hypothesize that the R. erythropolis mycothione reductase may form the basis of a cloneable tellurium nanoparticle (cTeNP). In the third chapter, metal(loid) reductase substrate specificity is developed through directed evolution of a glutathione reductase-like metalloid reductase (GRLMR). The native substrate of GRLMR is selenodiglutathione (GS-Se-SG), where zerovalent selenium nanoparticles are formed in the presence of NADPH. Error prone polymerase chain reaction was used to create a library of ~100,000 GRLMR variants. The library was expressed in Escherichia coli with 50 mM SeO32− to select a GRLMR variant with 2 mutations. One mutation (a D to E) appears to be silent, whereas the other (L to H) resides within 5Å of the active site. Compared to the GRLMR parent enzyme, the evolved enzyme became less capable of reducing reduced glutathione (GSSG) and GS-Se-SG in favor of SeO32−. The evolved enzyme also gained an ability to reduce SeO42−. We have described this enzyme as a selenium reductase (SeR). This is the first known instance of the substrate specificity profile of a metal(loid) reductase changing as a result of directed evolution. In the fourth chapter, the cNP concept is discussed in greater detail. The cNP synthesis paradigm is loosely defined as a system of ligands, reductants, and inorganic cations – where ligands are peptides, reductants are enzyme-cofactor pairs, and inorganic cations are dietary or supplemented metal(loid) ions. This modular platform is adaptable to a wide variety of metal(loid)/enzyme/peptide systems. The story of the creation of a cloneable Se nanoparticle (cSeNP) is also retraced. Briefly, a bacterial endophyte Pseudomonas moraviensis subsp. Stanleyae was found to be capable of efficient selenium reduction under aerobic conditions. Continued characterization led to the discovery of GRLMR which unraveled the cellular mechanism for reducing SeO32−. The enzyme can endow host cells with selenium resistance through nanoparticle formation when cloned. GRLMR was further modified through the fusion of a selenium nanoparticle-binding peptide which improved overall kinetic rates, nanoparticle retention, and nanoparticle uniformity. In the fifth chapter, preliminary work is described which may enable further development of the next generation of cNPs through reduced enzyme mass/mericity and 'multicolored' nanoparticles. Work is described which investigates the plasticity of GRLMR towards reducing other metals such as bismuth. Fluorescence assisted cell sorting (FACS) was used to determine if the relative quantity of intracellular metal(loid) nanoparticles can be differentiated, which is hypothesized to correlate to relative metal(loid) reductase activity. Whereas selenium content could be discerned between active and inactive GRLMR-expressing bacteria, relative bismuth content has yet to be analogously discerned. On the other hand, work was done towards rationally designing a monomeric GRLMR; there are ongoing efforts to use machine learning to graft the active site of GRLMR into a different monomeric template. Finally, a Muchor racemosus cytochrome b5 reductase (Cb5R) was identified in the literature which may serve as an ideal candidate to develop more minimalistic cSeNPs. Initial work has revealed that the enzyme is particularly resistant to soluble expression, which may hinder its ability to function as a clonable contrast agent. However, ongoing work is being done to 'supercharge' the enzyme to enable more facile expression.
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    ItemOpen Access
    Generation of site-specific ubiquitinated histones through chemical ligation and characterization of histone deubiquitinases
    (Colorado State University. Libraries, 2016) Al-afaleq, Nouf Omar, author; Yao, Tingting, advisor; Cohen, Bob, committee member; Fisk, Nick, committee member; Peersen, Olve, committee member
    Nucleosome is the basic unit of chromatin and is composed of 147 base pairs of DNA wrapped 1.65 turns around a histone octamer of the four core histones (H2A, H2B, H3 and H4)(Luger et al., 1997). Histones are subject to numerous post-translational modifications. One such modification is the addition of a single ubiquitin (Ub) moiety to a specific lysine residue in the histones, such as H2AK119 or H2BK120 in humans. Depending on the site of Ub attachment, these modifications have distinct functional consequences. Whereas H2A ubiquitination is associated with transcriptional repression and silencing, H2B ubiquitination is associated with actively transcribed regions and has roles in initiation, elongation and mRNA processing. A more recently discovered ubiquitination site in H2A, H2AK13/15, is associated with DNA damage repair. In addition, a number of other ubiquitination sites on all types of histones have been discovered by high throughput mass spectrometry. The functions and regulations of those novel ubiquitinations are not known. Deubiquitinating enzymes (DUBs) reverse these ubiquitinations and therefore, are involved in a variety of regulatory processes. Mutations in several histone DUBs have been implicated in various diseases, thus they represent potential therapeutic targets. The specificity and regulation of histone DUBs are poorly understood in part because it has been difficult to obtain homogenous ubiquitinated histones and nucleosomes to use as substrates in vitro. Previously, several strategies have been developed to produce chemically defined ubiquitinated histones that use a combination of expressed protein ligation (EPL) and solid phase peptide synthesis (SPPS) techniques. These protocols are technically challenging for a biochemical lab. This dissertation describes our successful approach in obtaining homogenous site-specific ubiquitinated H2A and H2B that were then reconstituted into nucleosomes and used to qualitatively and quantitatively characterize a panel of known histone DUBs in vitro. We anticipate that our approach can be applied to generate all types of Ub-histone conjugates regardless of the particular ubiquitination site or histone types. They will significantly facilitate the study of all types of histone ubiquitination.
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    Multi-color visualization and quantification of single RNA translation and HIV-1 programmed ribosomal frameshifting in living cells
    (Colorado State University. Libraries, 2019) Lyon, Kenneth Ray, Jr., author; Stasevich, Timothy, advisor; Munsky, Brian, committee member; Cohen, Bob, committee member; DeLuca, Jake, committee member
    Sixty years ago, Francis Crick first stated the central dogma of molecular biology: DNA makes RNA, RNA makes protein. At that time the central dogma could only be imagined, but over the past two decades revolutionary advances in fluorescence microscopy have now made it possible to directly image transcription and translation in living cells and organisms. A key breakthrough was the discovery and development of GFP, which can be genetically fused to other proteins to selectively light them up and track their expression in vivo. While this powerful technology can illuminate mature protein products in live cells, processes like translation remain in the dark. This is because fluorescent fusion tags take too long to mature and light up. By the time the fluorescence becomes visible, translation of a nascent protein is over and has long since separated from its parental RNA strand. This fundamental challenge has made it difficult to visualize, quantify, and study translational gene regulatory mechanisms in living cells and organisms with fluorescence microscopy. To achieve this, nascent chain tracking (NCT) was developed, a technique that uses multi-epitope tags and antibody-based fluorescent probes to visualize and quantify protein synthesis dynamics at the single-RNA level. NCT revealed an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides. By employing a multi-color probe strategy, NCT shows that a small fraction (~5%) form multi-RNA sites in which two distinct RNAs are translated simultaneously while spatially overlapping. NCT was then applied to further understand the dynamics of translating RNAs interactions with ribonucleoprotein (RNP) granules during cellular stress. NCT was used to image real-time single RNAs, their translational output, and RNA-granule interactions during stress. Although translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable, and sometimes rigid, associations with RNP granules. These stable associations increase with both mRNA length and granule size. Live and fixed-cell imaging demonstrated that mRNAs can extend beyond the protein surface of a stress granule, which may facilitate interactions between RNP granules. Thus, the recruitment of mRNPs to RNP granules involves dynamic, stable and extended interactions affected by translation status, mRNA length and granule size that collectively regulate RNP granule dynamics. Finally, NCT was utilized to quantify the dynamics of multiple open reading frames at the individual RNA level in a living system. An NCT multi-color imaging modality was used to investigate ribosomal frameshifting during translation. Frameshifts occur through a ribosomal +1(-2) or -1 (+2) nucleotide(s) "slip" into another frame and are implicated in both human disease and viral infections. While previous work has uncovered many mechanistic details about single-RNA frameshifting kinetics in vitro, very little is known about how single RNAs frameshift in living systems. Applying multi-frame NCT technology to RNA encoding the -1 programmed ribosomal frameshift (-1PRF) sequence of HIV-1 revealed that a small subset (~8%) of translating RNAs frameshift in living cells. This multi-color NCT method also revealed that frameshifting RNA are preferentially in multi-RNA sites, are translated at about the same rate as non-frameshifting RNA, and can continuously frameshift for more than four rounds of translation. Interestingly, fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide fresh insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.