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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2011)

Nitric oxide (NO) signaling in mammals occurs through activation of the soluble isoform of guanylate cyclase (sGC), which results in diverse physiological processes such as blood vessel dilation and neurotransmission. sGC is a heterodimeric heme protein that has evolved to be a specific sensor for NO. Although sGC contains the same histidyl-ligated porphyrin as the globins, it has no measureable affinity for oxygen and does not oxidize in air. These unusual features facilitate selective and robust activation by NO in aerobic cellular contexts. Questions regarding the mechanism of ligand discrimination in sGC led to the identification of sGC heme domain homologues in many different organisms where these proteins function as NO and/or oxygen sensors for gas-mediated signaling pathways. The members of the protein family have been subsequently termed Heme Nitric oxide/OXygen binding (H-NOX) domains due to their divergent ligand-binding properties. Functional characterization of H-NOX domains from prokaryotes has provided important clues about the structural features that control ligand discrimination across the H-NOX family. However, additional fundamental questions remain about the influence of protein structure on heme chemistry.

Extensive studies on the globins, as model heme proteins, have established the functional importance of heme protein topological features in modulating gas diffusion to and from the heme site. In globins, deep gas pockets around the porphyrin transiently capture and release oxygen, tuning oxygen affinity for transport, delivery, and storage in diverse physiological environments. Although mechanisms of gas binding in H-NOX proteins have been a subject of intense investigation, there is little knowledge regarding the functional role of protein structure in modulating gas diffusion. Using X-ray crystallography with xenon and kinetic measurements, a tunnel network that extends between the solvent and interior heme site was mapped in a prokaryotic H-NOX domain. Hindering gas diffusion through the tunnels has important consequences on diatomic gas affinity. This suggests that protein tunnels in H-NOX proteins may play functional roles in tuning gas-mediated signaling.

Unlike isolated H-NOX domains, mammalian sGC is a structurally more complex, multi-domain protein. The ability of the sGC heme to resist oxidation in air is unique among histidyl-ligated heme proteins and essential for maximal NO-induced activation. Speculations have been made about how the sGC heme resists oxidation, but no study has systemically addressed the structural and electronic factors that contribute to this critical property. To probe the accessibility of oxygen to the heme site, we sought to substitute the native heme of sGC with an unnatural porphyrin displaying emission that is quenched by oxygen. Using an expression-based methodology, a phosphorescent Ru porphyrin was incorporated in sGC constructs of varying lengths. Emission quenching results suggest that oxygen diffusion to the full-length sGC heme site is significantly hindered compared to smaller sGC constructs. Limited oxygen accessibility, combined with heme electronic factors, appear to serve as important evolutionary solutions in sGC to protect the heme cofactor under aerobic conditions.

Originally to probe sGC function, the strategies used to generate oxygen-sensing proteins have been applied to create a new family of H-NOX-based molecular sensors. Substitution of the native heme in bacterial H-NOX proteins with unnatural porphyrins has proven to be a promising strategy to develop stable protein agents for biological imaging applications. Heme proteins are highly tunable frameworks that coordinate porphyrins with high fidelity and specificity. These properties have been exploited to design heme protein-based sensors with tailored functionalities for enhancing porphyrin bioavailability. Initial work on making optical oxygen sensors has evolved into recent efforts to generate H-NOX-based MRI contrast agents with high relaxivities for future use in deep-tissue imaging. Thus, through substitution of the native heme group with unnatural porphyrins, new sensing properties have been built into the H-NOX family.

    Cover page: Tuning Sensing and Signaling in the H-NOX Family of Heme Proteins
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2020)
    An unusual feature of the opportunistic pathogen Candida albicans is its ability to switch stochastically between two distinct, heritable cell types called white and opaque. Here, we show that only opaque cells, in response to environmental signals, massively upregulate a specific group of secreted proteases and peptide transporters, allowing exceptionally efficient use of proteins as sources of nitrogen. We identify the specific proteases [members of the secreted aspartyl protease (SAP) family] needed for opaque cells to proliferate under these conditions, and we identify four transcriptional regulators of this specialized proteolysis and uptake program. We also show that, in mixed cultures, opaque cells enable white cells to also proliferate efficiently when proteins are the sole nitrogen source. Based on these observations, we suggest that one role of white-opaque switching is to create mixed populations where the different phenotypes derived from a single genome are shared between two distinct cell types.
      Cover page: An Opaque Cell-Specific Expression Program of Secreted Proteases and Transporters Allows Cell-Type Cooperation in Candida albicans
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2015)
      ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains. ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif. Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.
        Cover page: Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2017)
        The more than 500 protein kinases comprising the human kinome catalyze hundreds of thousands of phosphorylation events to regulate a diversity of cellular functions; however, the extended substrate specificity is still unknown for many of these kinases. We report here a method for quantitatively describing kinase substrate specificity using an unbiased peptide library-based approach with direct measurement of phosphorylation by tandem liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide sequencing (multiplex substrate profiling by mass spectrometry, MSP-MS). This method can be deployed with as low as 10 nM enzyme to determine activity against S/T/Y-containing peptides; additionally, label-free quantitation is used to ascertain catalytic efficiency values for individual peptide substrates in the multiplex assay. Using this approach we developed quantitative motifs for a selection of kinases from each branch of the kinome, with and without known substrates, highlighting the applicability of the method. The sensitivity of this approach is evidenced by its ability to detect phosphorylation events from nanogram quantities of immunoprecipitated material, which allows for wider applicability of this method. To increase the information content of the quantitative kinase motifs, a sublibrary approach was used to expand the testable sequence space within a peptide library of approximately 100 members for CDK1, CDK7, and CDK9. Kinetic analysis of the HIV-1 Tat (transactivator of transcription)-positive transcription elongation factor b (P-TEFb) interaction allowed for localization of the P-TEFb phosphorylation site as well as characterization of the stimulatory effect of Tat on P-TEFb catalytic efficiency.
          Cover page: Multiplex Substrate Profiling by Mass Spectrometry for Kinases as a Method for Revealing Quantitative Substrate Motifs
          • Article
          • Peer Reviewed
          Multicampus Research Programs and Initiatives (MRPI) Funded Publications (2015)
          ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide binding domains (NBDs)1,2. ABC exporters are present in both prokaryotes and eukaryotes with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases3,4. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus homologous to various multidrug transporters and containing one degenerate site with a non-catalytic residue next to the Walker B motif5. Here we report a subnanometer resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single particle electron cryomicroscopy (cryo-EM). The reconstructions clearly resolved characteristic features of ABC transporters, including helices in the transmembrane domain (TMD) and NBDs. A cavity in the TMD is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two NBDs remain in contact via their C-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two NBDs during the transition from the inward facing to outward facing conformations.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2018)
            Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.
              Cover page: Global substrate specificity profiling of post‐translational modifying enzymes
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2015)
              The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with (111)In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor-mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer-associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions.
                Cover page: Imaging Active Urokinase Plasminogen Activator in Prostate Cancer
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2018)
                Accurate predictions of T-cell epitopes would be useful for designing vaccines, immunotherapies for cancer and autoimmune diseases, and improved protein therapies. The humoral immune response involves uptake of antigens by antigen presenting cells (APCs), APC processing and presentation of peptides on MHC class II (pMHCII), and T-cell receptor (TCR) recognition of pMHCII complexes. Most in silico methods predict only peptide-MHCII binding, resulting in significant over-prediction of CD4 T-cell epitopes. We present a method, ITCell, for prediction of T-cell epitopes within an input protein antigen sequence for given MHCII and TCR sequences. The method integrates information about three stages of the immune response pathway: antigen cleavage, MHCII presentation, and TCR recognition. First, antigen cleavage sites are predicted based on the cleavage profiles of cathepsins S, B, and H. Second, for each 12-mer peptide in the antigen sequence we predict whether it will bind to a given MHCII, based on the scores of modeled peptide-MHCII complexes. Third, we predict whether or not any of the top scoring peptide-MHCII complexes can bind to a given TCR, based on the scores of modeled ternary peptide-MHCII-TCR complexes and the distribution of predicted cleavage sites. Our benchmarks consist of epitope predictions generated by this algorithm, checked against 20 peptide-MHCII-TCR crystal structures, as well as epitope predictions for four peptide-MHCII-TCR complexes with known epitopes and TCR sequences but without crystal structures. ITCell successfully identified the correct epitopes as one of the 20 top scoring peptides for 22 of 24 benchmark cases. To validate the method using a clinically relevant application, we utilized five factor VIII-specific TCR sequences from hemophilia A subjects who developed an immune response to factor VIII replacement therapy. The known HLA-DR1-restricted factor VIII epitope was among the six top-scoring factor VIII peptides predicted by ITCall to bind HLA-DR1 and all five TCRs. Our integrative approach is more accurate than current single-stage epitope prediction algorithms applied to the same benchmarks. It is freely available as a web server (http://salilab.org/itcell).
                  Cover page: Predicting CD4 T-cell epitopes based on antigen cleavage, MHCII presentation, and TCR recognition.
                  • Article
                  • Peer Reviewed
                  UC Merced Previously Published Works (2016)

                  Unlabelled

                  Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.

                  Importance

                  Biofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections.
                    Cover page: Global Identification of Biofilm-Specific Proteolysis in Candida albicans
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2017)
                    The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities. Differing iP specificity alters the quantity of production of certain MHC I epitopes but does not appear to be preferentially suited for antigen presentation. Furthermore, iP specificity was found to have likely arisen through genetic drift from the ancestral cP. Specificity differences were exploited to develop isoform-selective substrates. Cellular profiling using these substrates revealed that divergence in regulation of the iP balances its relative contribution to proteasome capacity in immune cells, resulting in selective recovery from inhibition. These findings have implications for iP-targeted therapeutic development.
                      Cover page: Immunoproteasome functions explained by divergence in cleavage specificity and regulation
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