Student Speaker Abstracts

Mechanistic Investigation of Polysaccharide Monooxygenases

Fungal polysaccharide monooxygenases (PMOs) oxidatively degrade cellulose and other carbohydrate polymers via a mononuclear copper active site using either O2 or H2O2 as a co-substrate. Cellulose-active fungal PMOs in the auxiliary activity 9 (AA9) family have a conserved second-sphere hydrogen-bonding network consisting of histidine, glutamine, and tyrosine residues. The second-sphere histidine has been hypothesized to play a role in proton transfer in the O2-dependent PMO reaction. Here the role of the second-sphere histidine (H157) in an AA9 PMO, MtPMO9E, was investigated. This PMO is active on soluble cello-oligosaccharides such as cellohexaose (Glc6), thus enabling kinetic analysis with the point variants H157A and H157Q. The variants appeared to fold similarly to the wild-type (WT) enzyme and yet exhibited weaker affinity toward Glc6 than WT. The variants had comparable oxidase (O2 reduction to H2O2) activity to WT at all pH values tested. Using O2 as a co-substrate, the variants were less active for Glc6 hydroxylation than WT, with H157A being the least active. Similarly, H157Q was competent for Glc6 hydroxylation with H2O2, but H157A was less active. Comparison of the crystal structures of H157Q and WT MtPMO9E reveals that a terminal heteroatom of Q157 overlays with Nε of H157. Altogether, the data suggest that H157 is not important for proton transfer, but support a role for H157 as a hydrogen-bond acceptor from a protonated dioxygen intermediate, thus facilitating catalysis with either O2 or H2O2.

Defining the role of C-terminal domain assembly and quality control in collagen associated disorders

Collagens are essential components of the extra-cellular matrix and basement membranes, where they serve to bolster tissue integrity, mediate cell migration and organize signaling. Collagen proteins have three domains: the C-terminal trimerization domain (CTD/NC1), the collagen domain and the 7S N-terminal domain. Collagens undergo folding and trimerization in the ER prior to secretion to the extra-cellular environment. This process requires a complex interplay between these domains. Mutations in collagens result in severe diseases, such as Osteogenesis Imperfecta, Alport’s Disease and Cerebral Small Vessel Disease which impair the integrity of bones, kidney and blood vessel in the brain and eye respectively. Although many disease-causing mutations in collagens have been identified, there still is an incomplete understanding of the mechanisms of pathogenesis. We hypothesize that CTD trimerization is a key step in determining the efficiency of collagen folding and secretion kinetics. To test this, we have designed and preliminarily tested a series of mutants of the COL4A2 CTD that are intended to either favor or disfavor assembly of the native a1(IV)2a2(IV)1 trimer. These mutations will be used as genetic tools to carefully probe the role of CTD folding and trimer assembly kinetics in ER retention, ER-associated degradation and secretion.

Massively parallel, single-molecule assessment of synthetic fidelity and drug-like properties in a DNA-encoded library

DNA-encoded libraries (DELs) have emerged as a promising drug discovery strategy, but successful translation of hits is often impeded by synthetic inefficiency and enrichment of poorly permeable compounds. Here we introduce a novel sequencing-based separation strategy, LC-seq, that simultaneously evaluates synthetic fidelity and permeability-relevant lipophilicity for individual DNA-encoded library members. Using a 120,000-member peptide library, we mapped reaction efficiency across all synthetic cycles and identified structure-reactivity trends. The on-DNA lipophilicities for resynthesized library members correlate strongly with their off-DNA lipophilicities and passive permeability in artificial membranes and MDCK cells. This approach enables direct assessment of compound quality and drug-like properties at unprecedented scale, potentially transforming DEL-based drug discovery.

Structurally Diverse Silyl Lipids to Modulate Lipid Nanoparticle Properties for mRNA Delivery

 Leah A. Patterson1, David A. Coppage1, Sydney M. Figueroa1, Angel A. Cobo1, Henriette O’Geen2, David J. Segal2,3, Annaliese K. Franz1*
1Department of Chemistry, University of California, Davis
2Genome Center, University of California, Davis
3Department of Biochemistry and Molecular Medicine, University of California, Davis

Abstract: Lipid nanoparticles (LNPs) are an emerging technology that has been widely used for therapeutic delivery, including nucleic acid delivery. LNPs contain amphiphilic lipids with polar head groups and nonpolar tails that can be structurally tuned to optimize delivery. Prior work in the field has involved modulating the head group to optimize delivery. Albeit more limited, examples of synthetic tail modifications also exist, such as branching and unsaturation. Branched and unsaturated tails are known to enhance mRNA delivery by reducing tail packing and increasing lipid fusogenicity. Furthermore, there are current limitations and new opportunities to control LNP properties related to stability, encapsulation and transfection. Here-in, we discuss the modular synthesis of a collection of novel cationic silyl lipids and their biophysical characterization and transfection as silyl-LNPs. We have demonstrated that the incorporation of silyl lipids with varying tail lengths, substituents, and silyldimethyl positions into LNPs controls liposome size, zeta potential, rRNA encapsulation efficiency (EE) and rate (ER), and mRNA transfection efficiency in HEK293T cells. Fifteen silyl lipids consistently demonstrated higher zeta potentials than all lipids evaluated including DOTAP (our model/control lipid), 5 demonstrated higher EE and ER, and 3 demonstrated higher mRNA transfection efficiencies in HEK293T cells. Of these top performing silyl lipids, several of them have shorter lipid tails with varying silyldimethyl positions and a phenyl or cyclohexyl ring. The three top performing silyl lipids from cell transfection have shorter tails, and two of them have the same tail structure (both containing a phenyl ring) but only differ in silyldimethyl position. This supports the importance of silyl position to control transfection efficiency. Our results also support that incorporation of the silyldimethyl group into the lipid tails increases liposome stability compared to lipids with a methylene in place of the silyldimethyl group. Future work involves the vivo testing of our top 3 silyl lipid candidates in Ai9 mice to assess their biodistribution, transfection efficiency and safety in vivo, as well as Cryo–TEM imaging, biophysical characterization, and biological testing of a new class of synthesized silyl lipids for mRNA and DNA delivery.

Monitoring monomer-specific acyl-tRNA levels in cells with PARTI

Abstract:  We describe a new assay that reports directly on the acylation state of a user-chosen tRNA in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non-α- amino acid monomers into protein: (1) the origins of the apparent selectivity of translation with respect to β2-hydroxy acid enantiomers; (2) the activity of PylRS variants for benzyl derivatives of malonic acid; and (3) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2’,3’-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non-α-amino acid monomers into proteins.

Potent Anticoagulant Discovered from a Traditional Chinese Medicinal Botanical: When Luck Meets Preparation

Traditional Chinese herbal theory was used to select a group of botanicals to screen for anticoagulant activity. From the active fractions, GNPS was used for dereplication. The generated molecular network was then used to determine potential active compounds. Closely related compounds were enzymatically produced and tested. A lipoxygenase product present in the extract of lycium chinense root bark was determined to be a potent inhibitor of collagen-induced platelet coagulation, selectively targeting the FcγRIIA receptor at nanomolar potency.

Mechanistic analysis of the [FeFe]-hydrogenase maturase HydE via Electron Paramagnetic Resonance and Native Mass Spectrometry

Liam P. Twomey1, Guodong Rao1, Casey J. Chen2, Thomas B. Rauchfuss3, Evan R. Williams2*, and R. David Britt2*

1Department of Chemistry, University of California, Davis

2Department of Chemistry, University of California, Berkeley

3Department of Chemistry, University of Illinois, Urbana-Champaign

Graduate Student; lptwomey@ucdavis.edu

The [FeFe] Hydrogenase enzyme uses an organometallic six-iron cofactor to interconvert protons and H2 rapidly at room temperature with minimum overpotential. This cofactor is constructed from a [Fe4S4] cluster joined via a proteinaceous cysteine to a unique organometallic [Fe2(adt)(CO)3(CN)2]2− cluster, termed "2FeH". The distal iron of this cluster is where catalysis occurs. This 2FeH cluster is synthesized from free Fe3+, cysteine, and tyrosine by the maturases HydE,F, and G with the assistance of the glycine cleavage system. The mechanism of HydG is known, and initial steps of the mechanism of HydE are understood, but the final product and method of product release from HydE are not known. The mechanism of HydF is also debated.

This work focuses on completing our understanding the mechanism of HydE by combining established organometallic semisynthesis methods with electron paramagnetic resonance (EPR) and native mass spectrometry (NMS). Using non-denaturing NMS of proteins allows the analysis of substrate binding, turnover, and product release in real time, as well as characterizing binding affinity and protein conformation as a function of the substrates. EPR provides complimentary information in the form of detailed electronic information at the active site.

We show here that all steps from the cleavage of the cysteinate ligand to dimerization to form the dithiolate dimer occur at HydE, raising questions about the function of HydF, which was previously assumed to be responsible for dimerization of the mononuclear precursor Complex B. This agrees with a recent computational study conducted with Prof. Lee-Ping Wang, which indicated condensation of HydE intermediates to the Fe(I) dimer. This also opens the system for further structural and biochemical analysis, and inspires speculation on the interaction modes between the HydA maturation and glycine cleavage systems.

Exploring 14-3-3/CRAF molecular glues in RASopathies

14‑3‑3 is a hub protein that interacts with hundreds of client proteins to regulate their activities. One 14-3-3 client is CRAF and binds to CRAF pS259 to prevent its activation in the MAPK pathway. The Arkin lab has developed molecular glues to selectively stabilize the 14-3-3/CRAFWT interaction and prevent the activation of CRAF by RAS. Mutations around S259 (CRAFNS) cause the RASopathy Noonan syndrome, resulting in an impaired interaction. Noonan mutations decreased the level of pS259 by 64-97% and decrease the binding affinity to 14-3-3 by 2- to over 12-fold compared to CRAFWT. Through modulation of protein-protein interactions (PPIs) with molecular glues, we can enhance the binding affinity of weakened interactions in disease. The 14-3-3/CRAFWT molecular glues stabilize the 14-3-3/CRAFNS interactions to varying degrees (2 to >100x) and strengthen the levels of pS259 up to 4.6-fold. A site-directed disulfide tethering screen has also been conducted to identify mutant-specific CRAFNS compounds. The results from this project will elucidate the biochemical and cellular effects of modulating weakened PPIs and could reveal a novel therapeutic strategy.

Programmable translational inhibition by a molecular glue-oligonucleotide conjugate

Selective inhibition of mRNA translation is a promising strategy for modulating the activity of disease-associated genes, yet achieving both high potency and specificity remains challenging. Rocaglamide A (RocA), a small molecule molecular glue, inhibits translation by clamping eIF4A1 onto polypurine tracts (PTs) found in many transcripts, limiting RocA’s specificity. Here, we developed RocASO, a chemical conjugate that links RocA to an antisense oligonucleotide (ASO) capable of base-pairing with defined mRNA sequences, thus directing RocA’s clamping mechanism to chosen targets and enhancing overall potency and specificity. We show that RocASOs are compatible with various types of ASO, including gapmers that degrade target RNAs. RocASOs were designed to effectively knock down endogenous genes (PTGES3, HSPA1B) and SARS-CoV-2 viral RNA, the latter conferring potent antiviral activity in cells. These findings establish RocASO as a versatile platform for programmable translational inhibition with therapeutic potential.