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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2023)

Biological containers known as protein cages are widespread in nature—displaying unique functionality via their ability to compartmentalize molecules in an internal lumen volume. Composed of repeated copies of a small number of protein subunits arranged symmetrically, protein cages are relatively simple in composition, and yet complex in structure. In nature these assemblies function in transport and protection, storage of important molecular cargo, and sequestration of metabolic reactions. These demonstrations of incredible functionality have inspired researchers seeking to engineer complex protein architectures with diverse functionality to direct their attention towards symmetric protein cages. Resulting from these efforts, a modest number of protein engineering studies have succeeded in replicating these structures de novo in hopes of applying them to the fields of medicine and biotechnology; however, developing applications for these assemblies that outperform existing technologies remains a challenging exercise. The primary aims of my thesis work are the design and development of applications for symmetric protein cages with a particular focus on mimicking natural functionalities not yet recreated via artificial protein design methods. We note the limitations of prevalent protein design methods, and technological advances that may revolutionize the field.The first chapter of this thesis is a brief introduction to protein cages as platforms for engineering, as well as efforts to design them de novo in the laboratory. In the second chapter of this thesis we sought to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. These cages incorporate a grafted interface onto each of two components making up the cages. In this work we describe challenges associated with direct rigid linkage of protein components via alpha helical extension and discuss whether the technique is a viable strategy for future cage design efforts. Next we attempt a novel approach to addressing a long-standing challenge in the field of protein design: engineering structurally responsive protein cages. The natural world holds numerous examples of protein cages that can assemble and disassemble in response to specific stimuli, but recreating this effect in the laboratory has proven challenging. In chapter three we present work on the design of protein cages that disassemble in response to a specifically chosen target protease. This work related to protein cage disassembly has implications in therapeutic design, as proteases are common therapeutic targets, and developing systems responsive to diseases biomarkers could be of great utility. In the next chapter we expand our focus on cage disassembly to other types of triggers for cage disassembly. In this work, we devise a system utilizing antibody-mimetic molecules called DARPins that allows for the design of protein cages that disassemble in response to any protein of interest. The work in this chapter focuses on a natural cage assembly (sulfur oxygenase) as a platform for development of this effect. The fifth chapter of this thesis covers work related to designing protein-cage based nanoparticles to be used as immunosorbents for patients on dialysis. The work addresses a clinical need for methods to remove the protein beta-2 microglobulin from the blood, as high concentrations are common in patients on long-term hemodialysis therapy, but can be toxic. We describe a protein cage that displays nanobodies on its exterior surface to generate beta-2 microglobulin adsorbent cages (BACs). In conclusion, the research performed in this dissertation work seeks to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. I then used previously described artificial protein cages to demonstrate triggerable protein cages with dynamic assembly properties that are responsive to molecular stimuli, as well as developing of an immunosorbent nanoparticle with clinical applications.

    Cover page: Design and Functionalization of Natural and Synthetic Protein Nanocages
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2024)
    Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.
      Cover page: AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2023)
      Beta-2 microglobulin (B2M) is an immune system protein that is found on the surface of all nucleated human cells. B2M is naturally shed from cell surfaces into the plasma, followed by renal excretion. In patients with impaired renal function, B2M will accumulate in organs and tissues leading to significantly reduced life expectancy and quality of life. While current hemodialysis methods have been successful in managing electrolyte as well as small and large molecule disturbances arising in chronic renal failure, they have shown only modest success in managing plasma levels of B2M and similar sized proteins, while sparing important proteins such as albumin. We describe a systematic protein design effort aimed at adding the ability to selectively remove specific, undesired waste proteins such as B2M from the plasma of chronic renal failure patients. A novel nanoparticle built using a tetrahedral protein assembly as a scaffold that presents 12 copies of a B2M-binding nanobody is described. The designed nanoparticle binds specifically to B2M through protein-protein interactions with nanomolar binding affinity (~4.2 nM). Notably, binding to the nanoparticle increases the effective size of B2M by over 50-fold, offering a potential selective avenue for separation based on size. We present data to support the potential utility of such a nanoparticle for removing B2M from plasma by either size-based filtration or by polyvalent binding to a stationary matrix under blood flow conditions. Such applications could address current shortcomings in the management of problematic mid-sized proteins in chronic renal failure patients.
        Cover page: Design of Beta-2 Microglobulin Adsorbent Protein Nanoparticles.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2019)
        We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB-Rifr system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics.
          Cover page: The Antibiotic Trimethoprim Displays Strong Mutagenic Synergy with 2-Aminopurine.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2022)
          Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.
            Cover page: Native Top-Down Mass Spectrometry with Collisionally Activated Dissociation Yields Higher-Order Structure Information for Protein Complexes
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2015)

            Aim

            To determine neuropsychological tests likely to predict cognitive decline.

            Methods

            A sample of nonconverters (n = 106) was compared with those who declined in cognitive status (n = 24). Significant univariate logistic regression prediction models were used to create multivariate logistic regression models to predict decline based on initial neuropsychological testing.

            Results

            Rey-Osterrieth Complex Figure Test (RCFT) Retention predicted conversion to mild cognitive impairment (MCI) while baseline Buschke Delay predicted conversion to Alzheimer's disease (AD). Due to group sample size differences, additional analyses were conducted using a subsample of demographically matched nonconverters. Analyses indicated RCFT Retention predicted conversion to MCI and AD, and Buschke Delay predicted conversion to AD.

            Conclusion

            Results suggest RCFT Retention and Buschke Delay may be useful in predicting cognitive decline.
              Cover page: Neuropsychological tests for predicting cognitive decline in older adults
              • Article
              • Peer Reviewed
              LBL Publications (2022)
              Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.
                Cover page: A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2013)
                The present study was undertaken to determine the expression and biological significance of HORMAD1 in human epithelial ovarian carcinoma. We found that a substantial proportion of human epithelial ovarian cancers expressed HORMAD1. In vitro, HORMAD1 siRNA enhanced docetaxel induced apoptosis and substantially reduced the invasive and migratory potential of ovarian cancer cells (2774). In vivo, HORMAD1 siRNA-DOPC treatment resulted in reduced tumor weight, which was further enhanced in combination with cisplatin. HORMAD1 gene silencing resulted in significantly reduced VEGF protein levels and microvessel density compared to controls. Our data suggest that HORMAD1 may be an important therapeutic target.
                  Cover page: Biological significance of HORMA domain containing protein 1 (HORMAD1) in epithelial ovarian carcinoma
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2020)

                  Background

                  Longevity as a phenotype entails living longer than average and typically includes living without chronic age-related diseases. Recently, several common genetic components to longevity have been identified. This study aims to identify additional genetic variants associated with longevity using unique and powerful analyses of pedigrees with a statistical excess of healthy elderly individuals identified in the Utah Population Database (UPDB).

                  Methods

                  From an existing biorepository of Utah pedigrees, six independent cousin pairs were selected from four extended pedigrees that exhibited an excess of healthy elderly individuals; whole exome sequencing (WES) was performed on two elderly individuals from each pedigree who were either first cousins or first cousins once removed. Rare (<.01 population frequency) variants shared by at least one elderly cousin pair in a region likely to be identical by descent were identified as candidates. Ingenuity Variant Analysis was used to prioritize putative causal variants based on quality control, frequency, and gain or loss of function. The variant frequency was compared in healthy cohorts and in an Alzheimer's disease cohort. Remaining variants were filtered based on their presence in genes reported to have an effect on the aging process, aging of cells, or the longevity process. Validation of these candidate variants included tests of segregation on other elderly relatives.

                  Results

                  Fifteen rare candidate genetic variants spanning 17 genes shared within cousins were identified as having passed prioritization criteria. Of those variants, six were present in genes that are known or predicted to affect the aging process: rs78408340 (PAM), rs112892337 (ZFAT), rs61737629 (ESPL1), rs141903485 (CEBPE), rs144369314 (UTP4), and rs61753103 (NUP88 and RABEP1). ESPL1 rs61737629 and CEBPE rs141903485 show additional evidence of segregation with longevity in expanded pedigree analyses (p-values = .001 and .0001, respectively).

                  Discussion

                  This unique pedigree analysis efficiently identified several novel rare candidate variants that may affect the aging process and added support to seven genes that likely contribute to longevity. Further analyses showed evidence for segregation for two rare variants, ESPL1 rs61737629 and CEBPE rs141903485, in the original longevity pedigrees in which they were initially observed. These candidate genes and variants warrant further investigation.
                    Cover page: Identification and genomic analysis of pedigrees with exceptional longevity identifies candidate rare variants
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2019)
                    Mycobacteriophages Deby, LaterM, LilPharaoh, Paola, SgtBeansprout, and Sulley were isolated from soil using Mycobacterium smegmatis mc2155. Genomic analysis indicated that they belong to subclusters K1 and K5. Their genomic architectures are typical of cluster K mycobacteriophages, with most variability occurring on the right end of the genome sequence.
                      Cover page: Genome Sequences of Cluster K Mycobacteriophages Deby, LaterM, LilPharaoh, Paola, SgtBeansprout, and Sulley
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