Thesis (M.S.)--University of North Carolina at Greensboro, 2007. / Title from PDF title page screen. Advisor: Gregory M. Raner; submitted to the Dept. of Chemistry. Includes bibliographical references (p. 78-80).

No / Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H2O2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme.

Heme is vital to a variety of cellular functions in bacteria ranging from energy generation to iron reserve. Group A streptococcus (GAS) is a prevalent bacterial pathogen that is responsible for an array of human diseases ranging from simple, self-limiting, mucosal and skin infections to invasive and systemic manifestations. GAS needs iron for growth and can satisfy this nutritional requirement by scavenging the metal from heme. The pathogen produces powerful hemolysins that facilitate heme release during infection. Heme is captured and relayed through the GAS cell wall and cytoplasmic membrane by dedicated receptors and transporters. To-date, the fate of the acquired heme is unknown in Streptococci. Although heme is nutritionally beneficial for GAS growth, its pro-oxidant and lipophilic nature makes it a liability with damaging effects on cellular components. The conundrum associated with heme use is particularly pertinent to GAS pathophysiology since invasive GAS infections involve massive hemolysis and the generation of unescorted heme in excess. In this dissertation, I aimed to describe the mechanisms that GAS uses for heme catabolism while managing its toxicity. I conducted a biochemical characterization of a new enzyme, HupZ in GAS that degrades heme in vitro. Similar to the heme oxygenase-1 (HO-1), HupZ activity leads to the formation of iron, CO, and a biliverdin-like product. I also investigated the impact of heme on GAS physiology and identified key mediators in the repair and detoxification process. This study demonstrated that heme exposure leads to a general stress response that involves the activation of antioxidant defense pathways to restore redox balance. Further, I studied a 3-gene cluster, pefRCD (porphyrin-regulated efflux RCD), which was activated by environmental heme, and provided support to my hypothesis that the pefRCD gene encodes a heme-sensing regulator (PefR) and heme efflux system (PefCD). I showed that the pef system protects GAS cells from heme-induced damage to the membrane and DNA by preventing cellular accumulation of heme. In conclusion, this dissertation addresses key knowledge gaps in GAS physiology and provides new insights into heme metabolism of GAS.

For pathogenic microorganisms, heme uptake and degradation is a critical mechanism for iron acquisition that enables multiplication and survival within hosts they invade. While the bacterial proteins involved in heme transport had been identified at the initiation of our investigation, the fate of heme once it reached the cytoplasm was largely uncharacterized. Here we report the first crystal structures of two members of the heme utilization operon from the human pathogen Escherichia coli O157:H7. These are the heme oxygenase ChuS in its apo and heme-complexed forms, and the apo form of heme binding protein ChuX. Surprisingly, despite minimal sequence similarity between the N- and C-terminal halves, the structure of ChuS is a structural repeat. Furthermore, the ChuS monomer forms a topology that is similar to the homodimeric structure of ChuX. Based on spectral analysis and carbon monoxide measurement by gas chromatography, we demonstrated that ChuS is a heme oxygenase, the first to be identified in any E. coli strain. We also show that ChuS coordinates heme in a unique fashion relative to other heme oxygenases, potentially contributing to its enhanced activity. As ChuS and ChuX share structural homology, we extended the structural insight gained in our analysis of ChuS to purport a hypothesis of heme binding for ChuX. Furthermore, we demonstrated that ChuX may serve to modulate cytoplasmic stores of heme by binding heme and transferring it to other hemoproteins such as ChuS. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuS and ChuX to probe heme binding sites and mechanisms in each. ChuS and ChuX mutants were analyzed through reconstitution experiments with heme and functional analyses, including enzyme catalysis by ChuS and mutants, and in culture development during heme challenge experiments by ChuX and mutants. Taken together, our results suggested that ChuX acts upstream of ChuS, and regulates heme uptake through ChuX-mediated heme binding and release. ChuS can degrade heme as a potential iron source or antioxidant, thereby contributing directly to E. coli O157:H7 pathogenesis. Functional implications that may be revealed from sequence and structure based information will be addressed as they pertained to our evaluation of ChuS and ChuX. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-04-27 11:34:50.272

Bacterial physiology and behavior is controlled by complex regulatory networks. Chemosensory systems are sophisticated signal transduction systems that can govern a range of cellular functions beyond that of traditional flagellar-based chemotaxis. The soil bacterium Myxococcus xanthus encodes eight chemosensory systems regulating multiple behaviors, including motility, exopolysaccharide production, and development. This work characterizes the Che7 system and demonstrates a role for Che7 in coupling aggregation and sporulation during multicellular development. The regulation requires an interaction between a single domain response regulator (CheY7) and a HEAT-repeat protein (Cpc7). A fatty acid desaturase, Des7, also impacts development in concert with the Che7 signaling system. Genetic analysis indicates the target of Che7 regulation is in the heme biosynthesis pathway, which is one aspect of iron homeostasis. Finally, characterization of iron and iron-responsive elements during development reveal a novel regulator, Fur2, that controls timing of development as well as che7 transcription. This work provides expands the known network regulating development in M. xanthus.

Streptococcus pyogenes (Group A Streptococcus) is a Gram-positive bacterial pathogen that causes significant superficial and invasive diseases. Iron acquisition is an important component of GAS pathogenesis in the human host. The 10 gene sia operon of GAS is involved in the acquisition of iron via heme or heme-binding proteins and encodes an ABC transporter as well as the large multifunctional receptor Shr. Domain analysis of Shr shows that it contains two copies of the DUF1533 (domain of unknown function) in its N-terminal part and two NEAT (NEAr Transporter) domains. NEAT domains are found in variable copy number in surface proteins of Gram-positive pathogens and are implicated in binding to various ligands. A new recombinant Shr protein was cloned and a purification protocol was developed, improving the yield of the full-length protein. A solid phase binding assay was developed and used to demonstrate Shr binding to hemoglobin. Several truncated Shr proteins were expressed and purified: the N-terminal Domain (NTD) up to but not including the first NEAT domain of Shr, the NTD plus the first NEAT domain (NTD-NEAT1) and the second NEAT domain alone (NEAT2). It was determined that Shr’s NTD mediates hemoglobin binding, demonstrating that a new protein pattern in Shr is involved in hemoglobin binding, and implicating the DUF1533 in this process. It was also determined that NTD-N1 and NEAT2 bind heme while NTD does not. Therefore, both NEAT domains may participate in the capture of heme from the host hemoglobin by Shr.

Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH), £\-melanocyte-stimulating hormone (£\-MSH), and £]-endorphin (£]-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to the hypoxia-inducible factor-1£\ (HIF-1£\) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the pro-survival MITF/HIF-1£\ pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1£\-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 £\ was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1£\ pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1£\/HO-1 indeed contributes to melanoma survival after POMC gene delivery.

Thesis (M. S. in Chemistry)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

Bacterial organisms continuously maintain homeostasis even in changing environments. This ability to maintain homeostasis is especially critical for pathogenic and opportunistic bacteria, which must adapt to both abiotic and biotic host environments. Both types of environments present unique limitations and conditions. Transition metal homeostasis under these varying conditions is important for bacterial survival. Transition metals such as zinc, cobalt, iron and copper are essential for cell survival, but become toxic if in excess. The host organism often takes advantage of this requirement by greatly limiting access to transition metals to limit infections, but in other environments, toxic levels of metal may be present. Bacterial organisms have developed many mechanisms to maintain transition metal homeostasis. This study focuses on two bacterial systems that are utilized to maintain metal balance; the heme-acquiring iron surface determinant (Isd) system of Bacillus anthracis and the copper and silver export Cus system of Escherichia coli. Host organisms use many proteins and systems to limit iron access from pathogenic bacteria, known as nutrient immunity. B. anthracis must acquire iron from the host organism upon infection and so has evolved multiple iron acquisition systems. The Isd system employs two extracellular proteins, IsdX1 and IsdX2, to remove heme from hemoglobin to use as an iron source. Once bound to heme, these hemophores transfer heme to a cell surface attached protein, IsdC, which further relays the molecule to be transferred into the cell for iron use. This study focused on the kinetics of heme transfer to better understand how acquisition occurs. This study determined that the oxidation state of the iron-heme molecule plays a significant role in the kinetics of heme acquisition by IsdX1 and subsequent transfer to IsdC. This work clarifies and further establishes the mechanism of iron acquisition by B. anthracis during infection. Copper and silver are used in many settings as antimicrobial agents, including as an alternative to antibiotic drugs. Pathogenic and opportunistic bacteria, such as E. coli, experience stress upon contact with copper and silver surfaces and materials. Copper is an essential transition metal, while silver is not biologically used, but both become toxic when in excess due to redox properties and disruption of biological molecules. E. coli utilizes several systems to remove excess copper and silver to resist toxicity. The Cus system, consisting of the soluble CusF and tripartite pump CusCBA, specifically exports copper and silver from the periplasm. Several roles of CusF have been suggested from in vitro data. The components CusAB were hypothesized to be the essential proteins of the CusCBA pump, while the outer membrane unit may not contribute specificity or be necessary for export. This study focused on the role and importance of CusF and outer membrane channel CusC during copper stress in vivo. An in vivo interaction between CusF and CusB was identified during copper stress. The data from this work indicate that cusF and cusC directly affect intracellular copper accumulation. Furthermore, this study revealed that SdsP may play in a secondary role to CusC to complement CusC to maintain copper resistance. This works establishes the importance of CusC as the main outer membrane component during copper export in E. coli.

No description available.