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Biochemical and Biophysical Characterization of Archaeal Intramembrane Aspartyl Proteases – Decoding Substrate Specificity, Kinetic Properties, and Solution Structure

(Georgia Institute of Technology, 2023-08-02) Wu, Yuqi

Intramembrane proteolysis is a conserved biochemical process, where the cleavage products are involved in diverse critical signaling events including cell differentiation, development, immune response and surveillance, and cholesterol metabolism. Conversely, IP dysfunction is often associated with human diseases. However, a complicated ternary complex composed of intramembrane protease (IP), transmembrane substrate, and lipid environment is always present and remains a major challenge in the study of IPs. Intramembrane aspartyl proteases, or IAPs, are least understood despite an increasing number of solved structures in recent years. In this work, we aim to understand IAP substrate specificity, kinetic properties, and solution structure through various biochemical and biophysical techniques and how lipid environment plays a role.
Molecular Details of Amyloid Formation by the Glaucoma-Associated Myocilin Olfactomedin (OLF) Domain

(Georgia Institute of Technology, 2023-04-17) Saccuzzo, Emily Grace

Primary open angle glaucoma (POAG) affects over 60 million people worldwide and is a leading cause of blindness in the US. Mutations primarily in the C-term olfactomedin (OLF) domain of myocilin account for ~5% of cases of POAG. It is hypothesized that mutant OLF undergoes a toxic gain of function that accelerates disease progression. Instead of being secreted, mutant myocilins are sequestered within the endoplasmic reticulum of trabecular meshwork (TM) cells, leading to cell stress and eventual cell death. TM cells are crucial for proper drainage of aqueous humor, and their premature death causes fluid buildup, increased intraocular pressure, and glaucoma-associated damage to the optic nerve. Preliminary data in our lab has implicated that aggregates formed by mutant OLF have hallmark of amyloid, a templated aggregate species that is highly resistant to degradation and is implicated in 50+ human diseases. Wild type (WT) OLF can also be driven to form amyloid when destabilized with increased temperature, chemical denaturants, or agitation. Study of model systems has revealed that for a folded protein to adopt amyloid, it must first undergo a transition into a partially unfolded state that is primed to aggregate into oligomers until a nucleating threshold is reached, at which point mature fibril formation ensues. This thesis provides the first molecular level insight into the mechanism by which destabilized OLF constructs undergo the transition into amyloid. A combination of NMR, kinetics and intracellular experiments revealed that destabilized OLF constructs are promoted to adopt a partially unfolded, aggregation prone state via unfolding from the inside out, and this unfolding is further propagated by loss of the internal calcium ion. To study the intermediate species in the amyloid formation pathway, a method was established for generating a cytotoxic oligomeric species that is on-pathway to fibril formation. Preliminary characterization of this construct with negative stain and cryogenic electron microscopy shows that this putative oligomer formed by destabilized OLF has a circular conformation with a central open cavity. Finally, for characterizing the end point mature fibrils, solid state NMR studies were done on fibrils formed by full length OLF. Preliminary 2D ssNMR experiments suggested that only a fraction of the 277 amino acids in the OLF sequence were involved in forming the fibril core. Unfortunately, the fibril sample used for ssNMR was heterogeneous and thus produced low resolution 3D spectra, which prevented full resonance assignments for the fibril core. However, the peptide “S-G-S-L” was assigned with relative confidence. This region only appears once in the OLF sequence, specifically in Peptide P1, a region previously identified via computational analysis and experimental studies to be relevant to promoting amyloid formation of full length OLF. Taken together, we now have molecular level insight into OLF amyloid formation, strengthening the relationship between myocilin-associated glaucoma and other amyloid diseases.
Development of recombinant antibodies for detecting multiple conformational states of glaucoma-associated myocilin

(Georgia Institute of Technology, 2023-04-12) Ma, Minh Thu

Throughout biomedical research, antibodies that recognize well-characterized epitopes are key reagents in the reproducibility and interpretation of immunoassays. Over the past three decades and after the COVID-19 pandemic, monoclonal antibodies have also emerged as a major class of successful clinical drugs. However, no such treatment currently exists for the debilitating eye disease glaucoma, which is the second leading cause of blindness worldwide. The heritable subtype of the most common type of glaucoma, POAG, is associated with the misfolding of the protein myocilin: mutant myocilin aggregates within the eye tissue trabecular meshwork, leading to cell death, severe damage to the optic nerve, and vision loss. Here, we develop recombinant, conformational antibodies that specifically target myocilin for research and therapeutics applications. These novel antibodies supersede existing commercial reagents due to their quality control and biological insight gleaned. Importantly, our antibodies also enable degradation of aggressive disease mutants of myocilin through cellular mechanisms afforded by their unique structural interactions with the protein. These exciting results provide proof of concept that targeted antibodies that ameliorate the insult of mutant myocilin aggregation could be therapeutic, and represent a new paradigm for treating other diseases associated with protein misfolding.
Expression, Purification, and Activity of Putative Intramembrane Aspartyl Proteases From Diverse Species

(Georgia Institute of Technology, 2022-04-27) Thomas, Gwendell Michelle

Intramembrane aspartyl proteases (IAPs) cleave peptide bonds within the hydrophobic lipid membrane. The best characterized IAP is presenilin, the catalytic subunit of γ-Secretase, which is known for cleaving amyloid precursor protein into the amyloid-β peptide that aggregates in the brains of Alzheimer’s patients. More than 100 substrates have been documented for γ-Secretase, yet no consensus recognition sequence has emerged, at least in part due to the technical complexities of studying this membrane-embedded proteolytic system. To date, the presenilin homolog from Methanoculleus marisnigri JR1 (MCMJR1) has been the sole microbial model of non-eukaryotic IAPs for in vitro molecular studies. A recent bioinformatic study uncovered over 1000 putative IAP sequences lurking in archaeal and bacterial organisms. Here, we report recombinant expression, purification, and enzymatic activity of selected new IAPs. These new IAPs express well and are from noted archaea such as H. volcanii and Lokiarchaeota. These putative IAP orthologs share key structural and cleavage preference similarities with MCMJR1 IAP and presenilin. By studying the molecular biochemistry of more IAP family members, additional trends and insights regarding cleavage preferences and peculiarities will emerge. Such knowledge will further illuminate the fascinating fundamental and complex chemistry occurring within the lipid membrane.
Contributions of active site residues to substrate binding and catalysis of 5-nitroanthranilic acid aminohydrolase

(Georgia Institute of Technology, 2019-04-30) Tao, Xingjian

Synthetic and naturally occurring nitroaromatic compounds are recalcitrant to degradation and they are toxic/mutagenic. The symbiont Bradyrhizobium sp. JS329 is the first microbe found to degrade a biological nitroaromatic compound, 5-nitroanthralinic acid (5NAA), which is secreted by the bacterium responsible for potato scabs, Streptomyces scabies. The first enzyme in the degradation pathway is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that has evolved to hydrolyze 5NAA to 5-nitrosalicylic acid. 5NAA-A is the first characterized metalloenzyme to utilize nucleophilic aromatic substitution. Here, I used isothermal titration calorimetry, enzyme activity assays, and X-ray crystallography to dissect contributions of individual active site and second-shell residues for assisting substrate transport. My studies demonstrate the interplay between substrate binding and catalysis requirements for this unusual metalloenzyme. Knowledge of the 5NAA-A structure and mechanism informs potential bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroaromatic and other challenging substrates.
Development of conformational myocilin antibodies, and biophysical characterisation of the mouse myocilin olfactomedin domain

(Georgia Institute of Technology, 2019-04-02) Patterson-Orazem, Athena Capucine

Mutations in myocilin are causative for the heritable form of open angle glaucoma in humans yet, almost 20 years after its discovery, the function of myocilin within the trabecular meshwork (TM) eye remains elusive. In this thesis, structural insights into the myocilin unique Y-shaped tetrameric architecture composed of N-terminal coiled coils and C-terminal olfactomedin (OLF) domain were employed to identify the target epitope and conformational specificity of popular myocilin-targeted antibodies. While available commercial antibodies target a range of structural domains, none were specific to natively folded myocilin. This prompted the development of new antibodies targeting folded epitopes across myocilin that are further cross-reactive for human and mouse proteins, to streamline reagent use in the laboratory. As part of the development of antibodies targeting the OLF domain, the crystal structure of mouse OLF was solved to 1.5 Å resolution. Further characterization of wild-type mouse OLF and mutants associated with glaucoma phenotypes in humans, revealed similar structure and stability between mouse and human OLF. However, aggregation kinetics for mouse OLF differ from humans, with implications for the relevance of mouse models for myocilin-associated glaucoma. For the N-terminal coiled coil regions, three new antibodies were developed. All three exhibit conformational selectivity and immunoprecipitate endogenous myocilin secreted from TM primary cells, and two are cross-reactive to both human and mouse myocilin, offering the opportunity to track folded myocilin in a variety of mouse and human tissues with a single reagent. These new antibodies offer the broader glaucoma research community the opportunity to track functional N-terminal myocilin in mouse and human samples, towards a deeper molecular understanding of both healthy and glaucomatous eye physiology.
Steps to Improving Stability of the β-propeller Structure of Myocilin's Olfactomedin Domain: Understanding the Evolution of the β-propeller

(Georgia Institute of Technology, 2017-05) Kwon, Michelle S.

Olfactomedin (OLF) domain-containing proteins, first identified in relation to bullfrog olfactory chemoreception, are part of a superfamily of proteins implicated in many important biological functions and human diseases. The myocilin OLF domain (mOLF), one of the best studied, is closely associated with the ocular disease glaucoma. Nearly 100 myocilin mutations have been reported in glaucoma patients; >90% are missense mutations within mOLF. Disease-associated mutant myocilins are destabilized and aggregation prone, leading to toxicity and death of cells that maintain the anatomical trabecular meshwork extracellular matrix in the eye. The Lieberman lab solved the crystal structures of OLF domains from myocilin and gliomedin (gOLF), a peripheral nervous system OLF domain. While both are similar five-bladed β-propellers, only mOLF contains a stabilizing calcium ion. Remarkably, gOLF is ~20 °C more stable than mOLF, even though it doesn't have a calcium ion and is phylogenetically more primitive. The goal of this project was to use insights from mOLF and gOLF to create a thermostable mOLF. Surprisingly, mutagenesis of a calcium-coordinating aspartate (D478) to alanine abolished calcium binding but increased mOLF thermal stability to near gOLF levels. Addition of D478A to the destabilized, glaucoma-associated variant D380A rescued thermal stability to that of wild-type (WT) mOLF. Structures of thermostable mOLF variants reveal unexpected changes in tertiary structure compared to WT mOLF, which were confirmed by solution biophysical measurements. The findings from this study expand our understanding of the structure-stability relationship of mOLF and provide further insight into the evolution of the OLF β-propeller.
Structural and functional characterization of an intramembrane peptidase and a non-peptidase homolog

(Georgia Institute of Technology, 2016-06-20) Kalyoncu, Sibel

Peptidases play fundamental roles in all living organisms and their dysfunction is associated with a variety of diseases. Although sequences of peptidases encoded in genomes throughout life have become readily available via high throughput sequencing technologies, research on their structural and functional characterizations lags behind due to challenges related to their crystallization and time-intensive biochemical/biophysical studies. Signal Peptide Peptidase (SPP) is an intramembrane aspartyl peptidase that cleaves signal peptides within the hydrophobic region of cellular membrane. SPP plays important roles in cellular functions such as immune system regulation. Structural characterization of membrane proteins including SPP is challenging due to their hydrophobic content which prevents crystallization. Structures of membrane proteins are severely underrepresented: number of unique membrane protein structures is still less than 1% in Protein Data Bank. Here, a new generalizable method was introduced to overcome crystallization challenge of membrane proteins. A toolbox of single chain antibody fragments (scFvs) specific to the EYMPME peptide (EE) epitope was developed for use as co-crystallization chaperones. Structures of all designed scFvs were solved and their crystallization propensities were systematically explored to improve their chaperone abilities. Tight complexation of anti-EE scFvs with EE-tagged SPP and another test membrane protein was demonstrated. Important lessons learned during crystallization and co-crystallization trials of scFvs and SPP are discussed in this dissertation. To understand peptidases at a mechanistic level requires both high resolution structures and extensive structure-function studies in which residues are systematically altered and differences in functionality of the peptidase are measured. Although a low resolution structure of inactive SPP became available during my PhD studies, details on how SPP recognizes and catalyzes its substrate are still not known. Here, preliminary data for a structure-function study to understand substrate gating mechanism of SPP are presented. Finally, structure-function studies of 5-nitroanthralinic acid aminohydrolase (5NAA-A), a metallo-peptidase family member that catalyzes a deamination reaction on a natural, toxic nitroaromatic compound, are presented. 5NAA-A has evolved a function other than peptide hydrolysis but is structurally and evolutionary related to peptidases and is thus classified as a non-peptidase homolog. We characterized 5NAA-A biochemically and biophysically, and obtained snapshots of its mechanism by solving its crystal structures in various states. The 5NAA-A structure and its nucleophilic aromatic substitution mechanism expand our understanding of the great diversity of enzymes capable of transforming natural organic compounds in our ecosystem.
Progress towards Co-crystalization of the E. coli Membrane Protein Intimin with Engineered Peptide-specific Antibody Fragments

(Georgia Institute of Technology, 2016-01-25) Heaner, David Prince

The determination of membrane protein structures is critical for the development of new pharmaceutical agents. Conventionally, membrane proteins are solubilized by the use of mild detergents. However, due to the lack of hydrophilic residues available to make crystal contacts and interference by the large detergent micelle, the quality of diffraction and resolution level needed for de novo structure determination is usually not obtained. In addition to the conventional detergent method, a new method using engineered single chain antibody fragments (scFv) and a Fab antibody fragment have been developed for use as crystallization chaperones. The scFv and Fab fragment interact with the membrane protein of interest via the EYMPME (EE) tag, which is selectively mutated into a hydrophilic loop of the protein. The membrane protein-antibody fragment complex may enter crystallization trials with the antibody fragment driving the complex nucleation through the formation of numerous, strong crystal contacts. Such a co-crystallization method with anti-EE scFv and Fab fragments provides the protein crystallographer with a “crystallization toolbox” that can be used for any crystallographic study of a protein of interest. Through size exclusion chromatography and SDS-PAGE analysis, complexation of the β-barrel membrane protein intimin harboring the EE tag with scFv/EE and Fab/EE has been shown to occur, from which crystallization trials have ensued. Formation of a co-crystal has proven to be difficult, which can be explained in part through molecular dynamics simulations of the mutated intimin L4 loop. This thesis work will present results and conclusions for this novel co-crystallization method.
Biophysical and structural characterization of proteins implicated in glaucoma and Gaucher disease

(Georgia Institute of Technology, 2011-08-24) Orwig, Susan D.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.