R-3 Repository :: Browsing by Author "Silberg, Jonathan"

Browsing by Author "Silberg, Jonathan"

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Bacterial Fragile Sites and Mutational Patterns of Human ‘Damageome’ Proteins
(2020-08-12) Mei, Qian; Silberg, Jonathan; Rosenberg, Susan M.
Genome instability fuels cancer, genetic diseases and evolution. Fragile sites are a source of genome instability, and are defined as regions of recurrent DNA damage in genomes. Although associated with many human diseases, the basis of fragility is still not fully understood. Holliday junctions (HJs) are four-way DNA junctions that form as intermediates in DNA repair and as a type of DNA damage when stalled replication forks collapse, and therefore are markers of fragility. Here, I identify three bacterial fragile sites of recurrent HJs located in the replication terminus (Ter) region of the Escherichia coli genome. I show that formation of HJs require DNA double-strand break (DSB) repair factors and are associated with recurrent DSBs, detected by DNA-end sequencing (END-seq). Two mechanisms that contribute to the three fragile sites are revealed. Due to the conserved nature of DNA transactions, my findings may help explain important disease-relevant unstable sites in human. In a separate study, the recently discovered DNA “damageome” proteins (DDPs) promote endogenous DNA damage when overproduced, and thus constitute another source of genome instability. Many DDPs and at least three DDP mechanisms have been found in E. coli using high-throughput methods. Despite the strong association of human DDP homologs with cancer-mutations, the study with experimental methods is more challenging. Here in this thesis, I mine public cancer databases to explore potential DDP roles in promoting mutations that drive cancers, to guide the experiments. Finally, the cancer chemotherapy drug 5-fluorouracil (5-FU) works by destabilizing the genome through the induction of thymineless death (TLD). We leverage the knowledge of E. coli TLD resistance genes to discover novel 5-FU-resistance genes in cancers.
Encryption of adeno-associated viruses with enzymatically decoded peptide locks
(2018-08-14) Judd, Justin; Suh, Junghae; Silberg, Jonathan; Rice University; United States Patent and Trademark Office
The present invention is a peptide lock that comprises at least one peptide that is genetically encoded into the Adeno-associated virus (AAV) capsid that block biologically active domains on the virus capsid surface. The peptide lock, can be processed by biological enzymes to restore biological behavior of the capsid-displayed domains, thus ‘decoding the lock’ or opening the lock. A method of forming the peptide lock comprises providing at least one peptide, providing an Adeno-associated virus capsid and genetically inserting the at least one peptide into the Adeno-associated virus capsid to block the biologically active domains on the virus capsid surface.
Frontiers in Programming Living Systems: Hybrid Cell-Material Systems for Real-Time Sensing and Actuating
(Rice University, 5/4/2024) Chappell, James; Frow, Emma; Kamat, Neha; Matthews, Kirstin; Montclare, Jin; Silberg, Jonathan; Sonkusale, Sameer; Tran, Lesa; Wilson, Corey
Investigations of the Specificity of Oxidosqualene Cyclization: Errors are the Rule, Not the Exception
(2014-12-05) Bodager, Paul Gregory; Matsuda, Seiichi P.T.; Ball, Zachary T; Silberg, Jonathan
This thesis describes the characterization of oxidosqualene cyclases from numerous organisms, through heterologous expression in Saccharomyces cerevisiae, and extraction from organismal tissue. Oxidosqualene cyclases are a family of proteins which catalyze the cyclization of the linear substrate oxidosqualene into cyclic compounds known as triterpene alcohols, acting in both the primary and secondary metabolism of organisms. Detailed analyses of cyclase product profiles in both primary and secondary metabolism are used herein to develop a comprehensive discussion of cyclase product specificity. First, the characterization of two oxidosqualene cyclases of secondary metabolism from the plant Arabidopsis thaliana, LUP4 and LUP5, by heterologous expression, is described. While demonstrating quite different product specificity, both cyclases make a mixture of nearly 20 triterpene alcohols. The isolation of a novel triterpene alcohol, (20S)-dammara-12,24-dienol, is reported. Next, the characterization of six oxidosqualene cyclases of primary metabolism are detailed, including lanosterol synthases from S. cerevisiae, Trypanosoma cruzi, Trypanosoma brucei, Homo sapiens, Bos taurus, and cycloartenol synthase from A. thaliana. Despite no reports of minor product generation by lanosterol synthases prior to this work, each cyclase is shown to make minor “errors”. These cyclases make different sets of minor products, and produce the major product with varying accuracy. This work demonstrates that minor product formation is characteristic of oxidosqualene cyclization, and leads to the conclusion that no cyclase produces only a single product. Finally, lanosterol synthase product profiles are extended to in vivo systems, via the analysis of triterpene alcohols present in yeast culture, as well as in mammalian tissue. This analysis demonstrates that S. cerevisiae lanosterol synthase produces at least 16 products, including three generated through B-ring-chair intermediates, the first evidence of a non-mutant cyclase accessing B-ring-boat and B-ring-chair intermediates. Analysis of bovine brain extracts led to the discovery that 18 lanosterol synthase minor products are detectable in mammalian tissue, including two novel triterpene alcohols, protosta-20(22)E-dienol and CB-thalianol A. Finally, this analysis demonstrated that one lanosterol synthase minor product, parkeol, is metabolized by enzymes in the sterol biosynthetic pathway, demonstrating that enzymatic errors generate a previously hidden level of chemical diversity in primary metabolism.
Monitoring iron-sulfur cluster content of human glutaredoxin 2 using fluorescence proteins
(2008) Li, Rui; Silberg, Jonathan
Current methods for directly determining whether an iron-sulfur cluster has been synthesized on a protein in vivo typically require that the protein of interest is purified prior to analysis for bound metallocluster. To establish an assay that can measure Fe/S-cluster synthesis in complex mixtures of biomolecules, we have fused a variety of fluorescent proteins (FP=BFP, CFP, GFP, and YFP) to human glutaredoxin 2 (Grx2) and characterized the effect of Grx2 metallocluster coordination on protein fluorescence. Gel filtration analysis revealed that FP-Grx2 fusion proteins are produced as a mixture of monomers and dimers when expressed in Escherichia coli , like native Grx2. The dimeric FP-Grx2 exhibited absorbance and circular dichroism spectra consistent with the presence of a Grx2-bound [2Fe2S] cluster, whereas the monomeric form of these fusion proteins lacked any detectable chromophores. Fluorescence analysis of the FP orthologs in these fusion proteins revealed that [2Fe2S]-cluster coordination produces a 30 to 50% reduction in FP fluorescence emission. These fluorescence indicators are expected to be useful for continuously monitoring Fe/S-cluster assembly on Grx2 in complex protein mixtures and should be useful for discovery of the proteins that mediate the assembly of metallocluster on Grx2 isoforms that are localized to both the nucleus and mitochondria.
Mutational Profiling of the Adeno-Associated Virus Rep Protein for Gene Therapy Production
(2024-10-24) Azim, Tasfia; Silberg, Jonathan; Thyer, Ross
Adeno-associated virus (AAV), a federally approved gene therapy vector that is currently in clinical trials for hundreds of diseases, often presents suboptimal characteristics for therapeutic applications, such as suboptimal tissue specificity, limited cargo size, undesired immune responses, and costly manufacturing. To improve this gene therapy vector, AAV proteins are frequently studied via rational design and combinatorial engineering. While the latter approach increases the sequence space that can be explored, it also presents unique challenges such as genotype-phenotype mismatches, noise arising from mutational errors in cloning, and bias arising in amplicon preparation and sequencing. In AAV, Rep proteins mediate DNA packaging and virus assembly, suggesting that changes in Rep activity, expression, or DNA binding might affect genome packaging. However, these proteins are not as well-understood as the proteins that make up the virus shell. I sought to understand how mutations in the Rep protein affect activity by selecting a library of Rep mutants for their ability to produce virions. To do this, I designed large protein libraries to examine a broad sequence space, I designed a selection strategy that couples genotype-phenotype characteristics of the mutants, and I designed a single-stranded DNA isolation workflow that enabled me to sequence winning variants in deep sequencing. By sequencing the rep gene following the purification of viruses that package AAV genomes, I identified Rep mutants having non-synonymous mutations with a range of cellular activities. Surprisingly, synonymous mutations within the p19 promoter were enriched to the greatest extent, increasing in abundance by 102 to 104-fold. When the most highly enriched mutant was used to package a synthetic DNA cargo into the AAV capsid, the packaging efficiency could not be differentiated from native Rep. These findings suggest that these synonymous mutations enhance AAV genome packaging into capsids by affecting Rep-genome interactions. They also suggest that silent sequence changes in the DNA cargo packaged by Rep can be used to tune packaging DNA packaging efficiency. Additionally, I designed a sequential cloning method for developing barcoded chimeric protein libraries, which enables easier analysis of deep-sequenced these. This cloning strategy has been partially validated. Future work should be done to optimize this cloning method, as it would be applicable for chimeric library design for any protein engineering experiment. Lastly, this work outlines considerations in high-throughput protein engineering experimental design. I hope this section of my thesis enlightens those who wish to begin high-throughput protein experiments and learn from some of the critiques I have of my own work.
Protein fragment complementation assay for thermophiles
(2012-08-07) Silberg, Jonathan; Nguyen, Peter Q.; Rice University; United States Patent and Trademark Office
A protein fragment complementation assay for thermophiles is provided wherein a thermophilic bacteria having a temperature-sensitive adenylate kinase is transformed with one or more vectors having sequences encoding a first test peptide operatively fused to a first portion of a thermostable adenylate and a second test peptide operatively fused to a second portion of the thermostable adenylate kinase. Association of the first and second test peptides allows association of the first and second portions of the thermostable adenylate kinase and growth of the thermophilic bacteria at a temperature greater than 70° C.
Regulating electron flow using fragmented proteins
(2022-08-09) Silberg, Jonathan; Atkinson, Joshua T.; Campbell, Ian J.; Bennett, George N.; Rice University; William Marsh Rice University; United States Patent and Trademark Office
Engineered protein electron carriers, microorganisms expressing the same, and methods detecting regulated electron flow are described.
The role of MK2/TTP pathway in regulating early development and innate immunity in zebrafish
(2018-11-30) Tandon, Bhavna; Silberg, Jonathan; Wagner, Daniel
The origin of novel structures and the genes that regulate their formation is a challenging question that has been difficult to address. One such novel structure is the yolk syncytial layer (YSL) in teleost fishes like zebrafish. I studied the genetic pathways operating in the YSL using the betty boop (bbp) mutant. betty boop encodes mitogen activated protein kinase activated protein kinase 2a (mapkapk2a), also known as mk2a. One of the downstream targets of MK2a is an RNA binding protein Tristetraprolin (TTP). bbp mutants lose the expression of YSL specific genes mxtx1 and mxtx2. Here, we show that these genes are regulated through their 3’untranslated region (UTR) by MK2a/TTP pathway required in the YSL during early zebrafish development to limit their expression to the YSL. I developed an in vivo reporter assay to detect spatial regulation of mRNA stability by the MK2/TTP pathway in early zebrafish embryos which can also be used to test other potential TTP targets. MK2a is the zebrafish homolog of mammalian MK2. In mammals, MK2 is not active during early development but rather is required to regulate genes that are responsible for inflammatory response. I show that MK2/TTP reporters are not regulated in early Xenopus embryo either, implying that the early requirement for MK2 arose after the division of tetrapod and teleost lineages. The role of mk2a and its gene duplicate mk2b in regulating immune response has not been previously studied in zebrafish. I developed zebrafish as a model infection system for infection by multiple species of Candida, an opportunistic pathogen and cause of an increasing number of hospital acquired infections. I developed novel transgenic zebrafish lines that allow inducible ablation of different phagocyte classes and identified different inflammatory responses to different Candida species. Preliminary results using these tools show that mk2b-/- mutants show a higher susceptibility to Candida hyphal infection, demonstrating that mk2b plays a role in innate immunity. Whether that role is the same that MK2 plays in mammals in regulating inflammatory cytokines still need to be determined. Taken together, all these findings suggest a model in which the ancestral MK2/TTP pathway was co-opted in teleosts to restrict expression of the mxtx genes to the yolk cell, but also retains its function in innate immune response.
Using methyl halides as a reporter in a model soil consortium and for intercellular signaling
(2023-08-08) Lu, Li Chieh; Silberg, Jonathan
Over the past decades, there has been an increasing understanding of the important and diverse role played by the soil microbiome in maintaining soil health, plant productivity, and biogeochemical cycles. Understanding the precise nature and contribution of these roles can allow us to harness soil microbial communities for agriculture, environmental engineering, and bioremediation. While -omics approaches and other bulk measurements have provided insights into soil microbial communities and their composition, these attempts can be confounded by a lack of suitable tools for understanding the perception and responses of individual microbes to different events and community members in soil. In this thesis, I review applications of synthetic biology to address some of the aforementioned challenges in researching and applying soil microbial communities. Then, I describe my work in evaluating and expanding the use of gas-output microbial biosensors in individual soil bacterial species and in a soil bacterial consortium via a gas-mediated cell-cell signaling relay. Additionally, I describe the construction of safe soil habitats for synthetic biology that allow us to evaluate intercellular microbial interactions and gas-output biosensor applications at centimeter- and meter-length scales. I also describe my efforts at constructing novel biosensor inputs for gas-output microbial biosensors and characterizing these new biosensors in liquid and soil environments. Finally, I discuss avenues for expanding on my work by applying gas-output microbial biosensors in more realistic soil conditions to answer fundamental soil science questions.

Browsing by Author "Silberg, Jonathan"

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