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Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Title
Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Creator
Raba, Daniel Alexander
Date
2019
Description
The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of...
Show moreThe proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of bacteria, including pathogenic species such as Vibrio cholera, Pseudomonas aeruginosa, Chlamydia trachomatis, as well as others. Residing in the cytoplasmic membrane of these bacteria, the enzyme couples the transfer of electrons to the pumping of cations across the cell membrane. In all previously studied homologues, the enzyme generates a sodium gradient through its pumping activity that can be utilized by the cell to power essential homeostatic processes. Furthermore, the electrochemical gradient generated by this enzyme has been shown to regulate the production of virulent factors and the efficacy of antibiotic extrusion and elimination. Although certain homologues have been investigated, particularly that of V. cholerae (Vc-NQR), the NQR homologues belonging to important pathogenic species have not been well studied. In the research detailed in this thesis, the first characterization of the NQR of P. aeruginosa (Pa-NQR) is described which identified this homologue as a new form of bacterial proton pump, differentiating it from all other studied homologues of NQR. Additionally, as part of this study our research group characterized the mechanism of inhibition of Pa-NQR by the molecule HQNO which is produced by P. aeruginosa and is known to be a strong inhibitor of Vc-NQR. Our results show that Pa-NQR possesses resistance to inhibition by this molecule compared to Vc-NQR, pinpointing residue F155 of subunit D as being important to resistance and the type of inhibition to be partial-mixed. Moreover, in further developing the understanding of the NQR of V. cholerae, we investigated the binding site of ubiquinone, the final electron acceptor of NQR’s electron transfer process, determining residues P185, L190, and F193 to be important for maintaining the structural composition of the ubiquinone pocket, ensuring efficient substrate binding and catalysis.
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Small Molecule Ligands and Antibody Drug Conjugates for Potential Application in Targeted Cancer Therapy and PET Imaging
Title
Small Molecule Ligands and Antibody Drug Conjugates for Potential Application in Targeted Cancer Therapy and PET Imaging
Creator
Ren, Siyuan
Date
2020
Description
Cancer has been a deadly disease threatening humans. Traditional treatment methods such as chemotherapy with non-specific targeting may cause...
Show moreCancer has been a deadly disease threatening humans. Traditional treatment methods such as chemotherapy with non-specific targeting may cause severe side effects to normal cells and tissues. Novel and better methods for treatment and imaging of cancer has been eagerly sought. We wanted to develop new small molecule inhibitors and antibody drug conjugates for targeted cancer therapy and imaging. Theranostic conjugates combining therapeutic entity and imaging agent are useful in treatment and diagnosis of cancer. We designed a theranostic conjugate containing iron chelating anti-tumor agent and optical imaging probe. Novel iron chelator CAB-NE3TA displayed significant anti-proliferation activity against several cancer cell lines and was conjugated to targeting antibody panitumumab (PAN). The therapeutic conjugate exhibited excellent anti-tumor efficacy and targeting ability to EGFR overexpressed on cancer cells such as skin cancer (A431). The theranostic conjugate CAB-NE3TA-PAN loaded with a near IR fluorescent dye provided promising anti-tumor efficacy and optical imaging in tumor-bearing mice. Tetrahydroisoquinoline (THIQ) analogues were synthesized and evaluated for cytotoxicity against different cancer cell lines. Our in vitro studies showed that the THIQ analogues exhibited anti-proliferative activities against multiple cancer cell lines. Cytotoxicity study revealed that anti-tumor activity of THIQ analogues are structure-dependent. Binding affinity between THIQ analogues and a potential target Tdp1 was determined. Positron emission tomography (PET) has been used in diagnosis of cancer. In this study, small molecules and chelators with potential donor groups were evaluated systematically for complexation with 89Zr for PET imaging. New ligands with different donors were evaluated for radiolabeling efficiency and complex stability with 89Zr. The theranostic conjugate (CAB-NE3TA-PAN-IR800) and small molecule THIQs and 89Zr-chelators showed encouraging results for potential applications in for therapy and imaging of cancer.
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Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Title
Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Creator
Fang, Xuan
Date
2019
Description
Cholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the...
Show moreCholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the antibiotic-resistance of its causative agent, V. cholerae. The resistance is mainly supported by a transmembrane electrochemical gradient of Na+ produced by the respiratory complex Na+-NQR coupled with an internal electron transfer pathway. The assembly and function of Na+-NQR is fulfilled by ApbE, the only known flavin transferase which covalently attaches two FMN molecules to the complex as part of its electron transport chain. Hence, ApbE is closely associated with the cause of antibiotic resistance. Because it does not have any human homologues, ApbE becomes an excellent drug target. In this work, we have investigated the physical properties of the enzyme and clarified its substrate specificity and pH dependence. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on the size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, pH dependence, mutagenesis, and steady-state kinetic studies have led us to identify the conserved His257 as a residue with dual roles: substrate binding and catalysis. Furthermore, bi-substrate kinetic studies have also revealed that ApbE follows a random Bi Bi mechanism. Together with structural studies, we propose a reaction mechanism where His257 functions as a base, shedding light into the understanding of the ApbE family.
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STRUCTURE AND DYNAMICS OF MODIFIED NUCLEOSOMES UNDER EPIGENETIC REGULATION
Title
STRUCTURE AND DYNAMICS OF MODIFIED NUCLEOSOMES UNDER EPIGENETIC REGULATION
Creator
Kohestani, Havva
Date
2022
Description
Epigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA...
Show moreEpigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA sequences. In vivo, reversible epigenetic mechanisms engage various molecular structures from RNAs to larger proteins. The present thesis investigates the influence of epigenetic regulatory factors such as histone protein variants and small non-coding RNAs on the dynamics and structure of nucleosome core particles. Our results show that a histone substitution is an efficient tool in increasing or decreasing the exposure of DNA to post-translational modification (PTMs) factors or larger molecular assembly elements. Substitution of canonical H2A with H2A.B alters DNA-dimer interface resulting in increased breathing and accessibility of DNA. Replacement of canonical H3 with CENP-A variant impacts the overall core-DNA dynamics with flexibility of DNA entry/ exit sites and more rigid tetramer structure. Histone substitution also affects the micro to macro level molecular communication in the nucleosome system. The long-range correlated motions are weakened in H2A.B compared to canonical NCP. We observed a reduction in effective long-range DNA-DNA and DNA-core allosteric pathways in CENP-A NCP compared to canonical and Widom NCPs. Non-coding RNAs increase the tendency of the H3 tail histones to interact with DNA and induce the structural changes in the initial ideal B-DNA of NCP. Overall, the interaction of epigenetic regulatory factors in the form of protein or nucleic acids shifts the energetic and structural properties of the original nucleosome system. As a result, the chromatin structure is prepared to generate the proper biological response throughout spermatogenesis, chromosome segregation, or PTMs assembly.
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COMPREHENSIVE ANALYSIS OF EXON SKIPPING EDITS WITHIN DYSTROPHIN D20:24 REGIONS
Title
COMPREHENSIVE ANALYSIS OF EXON SKIPPING EDITS WITHIN DYSTROPHIN D20:24 REGIONS
Creator
Niu, Xin
Date
2020
Description
Exon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to...
Show moreExon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to specifically mask specific exons and prevent them from being included in the mature mRNA. Exon skipping can also be used to restore protein expression in cases where a genetic frameshift mutation has occurred, and this how it is applied to Duchenne muscular dystrophy, DMD. DMD most commonly arises as a result of large exonic deletions that juxtapose flanking exons of incompatible reading frame, which abolishes dystrophin protein expression. This loss leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is to restore the reading frame by skipping an exon adjacent to the patient’s original. While restoring some protein expression is good, how removing some region from the middle of protein affects its structure and function is unclear. Complicating this in this case is that the dystrophin gene is very large, containing 79 exons. Many different underlying deletions are knowns, and exon skipping can be applied in many ways. It has previously been shown that many exon-skip edits result in structural perturbations of varying degrees. Very few studies are focused on the protein biophysical study and it is still basically unclear whether and how such editing can be done to minimize such perturbations. In order to provide the solid evidences which prove the significant variation among those cases (especially for the clinically relevant cases) and better understanding the general principles of “what makes a good edit”, we examine a systematic and comprehensive panel of possible exon edits in a region of the dystrophin protein. The domain D20:24 of dystrophin rod region are selected for its entirety which is separated by hinge region (mostly random coiled structure) and addition of other STRs will not disrupt the structure stability. Also D20:24 regions lie in the Hot Spot region II (HS2) which holds the most number of DMD patients. During the comprehensive scan, we identify for the first time, exon edits that appear to maintain structural stability similar to wild-type protein and those clinically relevant edits. Then we figure out the factors that appear to be correlated with the degree of structural perturbation, such as the number of cooperative protein domains, as well as how the edited exon structure interacts with the protein domain structure. Our study is the first systematic and comprehensive scan for an entire multiple STRs domain. This would help us understand the protein nature of various exon skipping edits and provide useful target for clinical treatment. Also the knowledge we learned may be applied to produce more sophisticated CRISPR edits in the future work.
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UTILIZING BACTERIAL INTERACTIONS TO CONTROL PATHOGENIC BIOFILM FORMATION
Title
UTILIZING BACTERIAL INTERACTIONS TO CONTROL PATHOGENIC BIOFILM FORMATION
Creator
Fang, Kuili
Date
2020
Description
Many chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm...
Show moreMany chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm formation is a result of dynamic intra- or inter-species interactions. However, the nature of molecular interactions between bacteria in multi-species biofilms are not well understood compared to those in mono-species biofilms. The first project (Chapter 3) investigated the ability of probiotic Escherichia coli Nissle 1917 (EcN) to outcompete the biofilm formation of pathogens including enterohemorrhagic E. coli (EHEC), Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis. When dual-species biofilms were formed, EcN inhibited the EHEC biofilm population by 14-fold compared to EHEC mono-species biofilms. This figure was 1,100-fold for S. aureus and 8,300-fold for S. epidermidis; however, EcN did not inhibit P. aeruginosa biofilms. In contrast, commensal E. coli did not exhibit any inhibitory effect toward other bacterial biofilms. We identified that EcN secretes DegP, a bifunctional (protease and chaperone) periplasmic protein, outside the cells and controls other biofilms. Although three E. coli strains tested in this study expressed degP, only the EcN strain secreted DegP outside the cells. The deletion of degP disabled the activity of EcN in inhibiting EHEC biofilms, and purified DegP directly repressed EHEC biofilm formation. Hence, probiotic E. coli outcompetes pathogenic biofilms via extracellular DegP activity during dual-species biofilm formation. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a pathogen causing the outbreaks of hemorrhagic colitis. Conventional antibiotics treatment is not recommended for EHEC infection as antibiotics trigger Shiga toxin production of EHEC and aggravate hemolytic-uremic syndrome. EHEC biofilm formation is closely associated with its virulence expression. Previously, we identified that probiotic E. coli Nissle 1917 (EcN) secretes DegP resulting in the inhibition of EHEC biofilm formation in a dual culture. DegP is a serine protease exhibiting both proteolytic and chaperone functions and binds to outer membrane proteins (OMPs) of target cells. However, the extracellular function of DegP is not clear. We hypothesized that binding of DegP to OMPs of EHEC might inhibit EHEC biofilm formation by affecting the adhesion ability or changing biofilm-related gene regulations of EHEC. We constructed EHEC mutants lacking ompA, ompC, or ompF individually and in combination and assessed their biofilm formation in the presence of DegP-secreting EcN in the co-culture or by adding purified DegP. It was found that both ompA and ompC double deletion decreased EHEC single species biofilm, and also caused that DegP inhibited more EHEC biofilm (about 25 fold inhibition) than DegP inhibited EHEC wt biofilm (about 10 fold), indicating that OmpA and OmpC are more related to EHEC biofilm than OmpF, and OmpA and OmpC might deplete DegP inhibitory functions. On the other hand, DegP S210A, a DegP mutant lacking protease function, inhibited EHEC wt biofilm, indicating that DegP’s biofilm inhibition function is not from its protease activity. Additionally, EHEC transcription profiles in the presence of DegP showed that DegP up-regulated expressions of cellulose production related genes (csgD and bcsA) and motility related genes (flhD, qseB), which were all involved in EHEC biofilm inhibition, and down-regulated Shiga toxin 2 virulence gene (stx2). Besdies, DegP promoted EHEC cellulose production and motility, which is consistent with transcription profile, and Shiga toxin 2 production will be further tested. This study reveals a new function of DegP secreted by EcN in controlling biofilms and leads us to develop an alternative strategy to control biofilm-related infections. Foodborne pathogen Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the Chapter 5 study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 times 10^6 CFU/cm2 L. monocytogenes ATCC19115 in single-species biofilms to 1.1 times 10^5 CFU/cm2 in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (> 30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.
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The role of fibrillar collagen in tissue function
Title
The role of fibrillar collagen in tissue function
Creator
Ma, Yin
Date
2020
Description
Fibrillar collagen plays an important role in maintaining soft tissue integrity and providing chemical and physical cues for cell fate...
Show moreFibrillar collagen plays an important role in maintaining soft tissue integrity and providing chemical and physical cues for cell fate decisions. Collagen remodeling, which alternates the amount, distribution, and biomechanics of collagen, primarily type I (COLI) and type III (COLIII), can change tissue properties. This process is essential not only in biological developments but also in pathological processes. Thus, it is meaningful to understand the correlation between collagen remodeling and tissue dysfunction and investigate the cells' response to fibrous protein matrices. However, current studies in biochemical analysis of collagen and biomechanical study of tissues were carried out at different scales. So it is hard to correlate the data to draw solid conclusions. In this thesis research, we used two collagen disorder associated pathological conditions, pelvic organ prolapse (POP) and micropapillary serous carcinoma (MPSC) of the fallopian tube, as models to unravel the correlation between tissue dysfunctions and the impaired microenvironment relevant to the composition, nanostructure, and biomechanics of a collagen fibril. In the case of POP, we found the collagen fibers in tissues of POP patients were less abundant but stiffer than those of non-POP individuals, implying a loose and fragile matrix that is weakly integrated with other components of the connective tissue to provide adequate support of the pelvic organs. On the other hand, the collagen D-period, the characteristic banding feature which signals the proper assembly of collagen molecules, decreased in POP tissues. We surmised that the molecular level changes of collagen in POP were accountable for the weak matrix mechanics, verified by a systematic in vitro study. We also examined the collagen matrix alternation in MPSC of the fallopian tube, which is thought to cause ovarian cancer via metastasis. Since cancer metastasis is often related to collagen remodeling, we examined the collagen matrix alternation in this disease. We observed the heterogeneous distribution of COLI and COLIII in the papillae of the tumor tissue. Noticeably, COLI was accumulated at the papillae tip, whereas COLIII was dominant at the papillae base. We also observed the absence of collagen matrix between the micropapillary tip and the fibrosis base. Such an uneven collagen distribution implies that the matrix exerted distinctive forces on the tumor cells to regulate their behaviors, including cell migration, directional growth, and shedding from the primary tumor to initiate metastasis. These conclusions have been supported by the results of our in vitro experiments. In investigating the effect of the microenvironment on cell behavior, we established and validated an AFM-based method to collect and quantitatively analyze the mRNA samples from targeted live cells at the single-cell level. This method overcomes issues, such as severe cell damage or even cell death, the capability of time-dependent and in situ analyses, in current methods. The application of the method in studying heterogeneous gene expression in single cells and the interaction between cancer cells and cancer-associated fibroblasts was demonstrated. We also demonstrated that this method can be potentially used to quantitatively analyze the gene expression level changes in a targeted cell in response to the microenvironment.
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