Chemical and Biomolecular Engineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2751
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Item HISTATIN 5 MODIFICATIONS IMPACT PROTEOLYTIC STABILITY IN THE PRESENCE OF FUNGAL AND SALIVARY PROTEASES(2024) Makambi, Wright Kingi; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Candida albicans, found in the oral cavities of 30-50% of the global population, can lead to oral candidiasis, particularly in immunocompromised individuals like those with HIV or diabetes. The current treatments, small-molecule antifungals, often fall short due to drug resistance and toxicity. To address these challenges, histatin 5 (Hst5), a 24-amino-acid peptide naturally present in human saliva, has been studied as a potential antifungal therapy. Hst5, however, is susceptible to degradation by secreted aspartyl proteases (Saps) produced by C. albicans and salivary enzymes, limiting its potential efficacy as a therapeutic. We have engineered Hst5 variants utilizing rational design in order to understand the interactions with Saps and Saliva. We have also made advancements in developing a novel screening method utilizing the directed evolution technique yeast surface display. Our study employed rational design to modify Hst5, at its lysine residues (K5, K11, K13, and K17), substituting them with leucine or arginine to examine their influence on interactions with Saps (Sap1, Sap2, Sap3, Sap5, Sap6, Sap9, and Sap10). Sap5, Sap6, and Sap10 did not degrade Hst5 at the tested conditions, while Sap1, Sap2, Sap3, and Sap9 did. Some modifications, such as K13L, are particularly susceptible to proteolysis by Sap1, Sap2, Sap3, and Sap9. In contrast, K17L substantially increases the stability and antifungal activity of Hst5 in the presence of Saps. Additionally, although the K11RK17L variant was degraded more than the K17L variant, their antifungal activities were largely similar. The proteolysis products of were also identified by mass spectrometry identifying the [4-24], [1-17], and [14-24] Sap proteolysis products. We also evaluated the proteolytic stability of these variants in saliva. Both K17L and K5R showed improved stability; however, the enhancements were modest, suggesting that further engineering is required to achieve significant improvements. Further experiments evaluated how additional amino acid substitutions at K13 and K17 affect the peptide’s proteolytic stability in the presence of Saps (with and without zinc). Our findings suggest that the positive charge at K13 is important for the proteolytic stability of Hst5, as all other variants tested except K13R reduce overall proteolytic stability. Furthermore, many substitutions at K17, including tryptophan, significantly enhance proteolytic resistance and antifungal activity following incubation with Saps. The K17W variant showed improved stability and antifungal efficacy, maintaining its function even in the presence of zinc and exhibiting stronger antibiofilm activity than the parent Hst5. In addition to the rational design work, we have advanced the development of a directed evolution yeast surface display platform for screening peptides for proteolytic stability. This would allow for the expression of large peptide libraries on the surface of Saccharomyces cerevisiae. Through optimization of expression and display conditions, we determined an induction media at 30°C with a pH of 3.5 and devoid of glucose improved the expression and display of Hst5 peptides on the surface of S. cerevisiae. We also optimized the degradation conditions for Sap2 37°C, a pH not exceeding 7.4, and a Sap2 concentration of 0.78 µg/mL led to the best discrepancy between proteolytically stable variants. Additionally, we found that a 40 amino acid linker between the peptide and the yeast surface provided the best observing proteolytic degradation. Using the optimized system, we showed that yeast surface display can be used to discriminate between peptide variants with different levels of proteolytic stability. This lays the foundation for future work to screen large libraries of peptides for proteolytic stability. From these results, we have gained a deeper understanding of the interactions between Hst5 and Saps, showing that modification at different lysine residues greatly impacts the proteolytic stability of Hst5. Furthermore, we have shown that the yeast surface display platform can be used to screen the proteolytic stability of peptides. Looking forward, this peptide should be engineered for proteolytic stability in saliva. Furthermore, mock screens should be made before screening a library of peptides using the yeast surface display platform.Item EXPERIMENTAL INVESTIGATION OF THE LIPID-BINDING MECHANISM OF OSH4 PROTEIN(2024) Konakbayeva, Dinara; Karlsson, Amy; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent findings show that intracellular lipid traffic between organelles primarily occurs through a non-vesicular pathway involving lipid transport proteins (LTPs) and is facilitated by areas of close apposition between two organelles so called membrane contact sites (MCS). Oxysterol-binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae serve as examples of LTPs. Osh proteins are crucial for transporting signaling lipids and are believed to form MCSs. In this study, we examined the binding mechanism of the Osh4 protein, aiming to gain a better understanding of its explicit membrane-binding mechanism.The Osh4 protein possesses an α-helical binding domain known as the amphipathic lipid-packing sensor (ALPS)-like motif. Our approach involved utilizing experimental methods to examine the biophysical interactions of both the ALPS peptide and the full-length Osh4 protein. To investigate the binding interactions of ALPS with membranes of different lipid compositions, we examined its interactions with three different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; has a zwitterionic head group) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS; has a negatively charged head group)—1:1 POPC-POPS, 4:1 POPC-POPS, and 9:1 POPC-POPS—as well as pure POPC. To understand the structural changes in ALPS and model membranes during peptide-membrane interactions, we performed a series of experimental studies. Circular dichroism (CD) was used to study the changes in the secondary structure of ALPS in different environments. The CD data indicated that the α-helical conformation of the ALPS peptide was more pronounced in the presence of POPC-POPS liposomes, especially with a higher content of POPS lipid, compared to liposomes composed entirely of POPC. This observation underscores the significant influence of anionic lipids in the facilitation of peptide folding at the membrane-water interface. X-ray diffraction was utilized to study the changes in membrane structure upon ALPS binds to it. The X-ray diffraction results showed that the ALPS peptide caused thinning of the multilayer with an increased POPS lipid ratio. This could be due to the electrostatic interaction of the positively charged Lys residue in the ALPS sequence with the anionic POPS lipid. We also studied the binding of the peptide to membranes by observing changes in the Trp fluorescence emission spectrum of ALPS upon the addition of liposomes. We observed a blue shift in the fluorescence emission maximum of Trp with higher POPS content. This suggests that the ALPS peptide was experiencing a more hydrophobic and less polar environment in the presence of the liposomes, indicating possible penetration of the peptide into the hydrocarbon region of the bilayer. The blue shifts of Trp emission in the presence of POPS liposomes were higher than those observed with POPC liposomes and suggest that the ALPS peptide binds better to charged POPS lipids, which is consistent with the X-ray diffraction data. We also conducted Trp fluorescence titration and ITC experiments to gain deeper insights into the binding affinity of the ALPS peptide to a model membrane. Using fluorescence data, we estimated the binding constant for the binding of ALPS to liposomes by performing titration measurements of vesicles with the ALPS peptide. Our analysis demonstrated that ALPS binding to 4:1 POPC-POPS lipid membranes had a Kd of 1.88 ± 0.47 μM, which corresponds to a free energy change (ΔG) of -7.82 ± 0.15 kcal/mol. Additionally, the ITC experiments performed with the same vesicles yielded a ΔG of -4.41± 0.04 kcal/mol. This result is slightly less than the ΔG value of -7.82 ± 0.15 kcal/mol obtained from fluorescence spectroscopy titration. The observed discrepancy of -3.41 kcal/mol may indicate the energy associated with the folding of the ALPS peptide. In order to understand how Osh4 forms MCSs between two membranes, we need to examine how the membranes interact with the full-length protein. The first step to achieve this is to produce the protein through recombinant protein production methods. After evaluating two different fusion tags, glutathione S-transferase (GST) and small ubiquitin-related modifier (SUMO), it was found that the SUMO tag resulted in higher protein yield and greater protein purity. Our work lays the foundation for future experiments with the full-length Osh4 protein to improve our understanding of the mechanisms of lipid transport between membranes. Our results emphasize the ALPS peptide’s selectivity for specific lipid environments, particularly its affinity for anionic lipids. We demonstrated that the presence of anionic lipids is crucial for the motif's ability to induce conformational changes upon binding to a membrane, and these conformational changes likely play a critical role in intracellular lipid trafficking and membrane organization.Item Engineering cell-penetrating peptides for translocation and intracellular cargo delivery in Candida species(2017) Gong, Zifan; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal infections caused by Candida species, particularly C. albicans and C. glabrata, have become a serious threat to public health. The rising drug resistance has prevented effective treatment and increased the mortal rate. Novel approaches to improve the therapeutic effects of antifungal agents and allow delivery of agents that are not normally cell-permeable are in demand. In order to improve the intracellular delivery of antifungal agents, we have investigated using cell-penetrating peptides as drug carriers for treating fungal infections. CPPs have been widely studied as tools for delivering a variety of molecular cargo into cells, including DNA, RNA, proteins, and nanoparticles. Previous work with CPPs has mainly focused on their uptake in mammalian cells, but CPPs also have potential as drug delivery and research tools in other organisms, including Candida pathogens. We have explored various well-studied CPPs to identify peptides that retain their translocation capability with Candida cells, including pVEC, penetratin, MAP, MPG, SynB, TP-10 and cecropin B. The CPPs pVEC, penetratin, MAP and cecropin B show a higher level in the cytosol adopt direct translocation mechanisms and exhibit toxicity towards C. albicans. Our peptide localization and mechanistic studies allow better understanding of the mode of translocation for different CPPs, which is related to the potential toxicity towards Candida pathogens. To further understand the molecular mechanisms of translocation of CPP, we investigated the biophysical properties of the peptides. CPPs that previously were shown to use direct translocation mechanisms (pVEC, MAP, and cecropin B) exhibit helical conformations upon interaction with cells due to the hydrophobic interaction with the core of bilayers. Membrane associations of peptides that entered cells via endocytosis were controlled by electrostatic forces. Our novel structure characterization methods using circular dichroism with live fungal cells, along with Monte Carlo simulations, allow us to understand how CPPs interact with cell membranes and how the membrane association affects the translocation mechanisms. After beginning to understand the structure-function relationships of CPPs, we engineered two CPPs, pVEC and SynB, to enable better translocation efficacy and manipulation of translocation mechanisms. We tuned the properties of the peptides, including the net charge and the hydrophobicity, to alter intracellular fates and the level of antifungal activity. These results are promising and motivate better peptide engineering for specific purposes. Our work with CPPs and fungal pathogens contributes to the understanding of structure-function relationship of CPPs in Candida species. We have provided the foundation for further peptide engineering and explorations into applications of CPPs in treating fungal infections.