NMR Structural Studies of Type III Secretion System Tip Proteins

The type III secretion system (T3SS) is a complex set of regulatory and structural protein machinery common to many Gram-negative bacteria for virulence. Many of these bacterial species are human pathogens and cause a variety of infectious diseases. These resulting diseases can be fatal and range from chronic infections of the lungs in cystic fibrosis patients from Pseudomonas aeruginosa infection to gastroenteritis from Salmonella typhimurium. Each of these species uses the T3SS to deliver bacterial effector proteins into the host cell cytosol in order to manipulate normal host cell functions. The purpose of these host cell alterations varies widely between bacterial species, including prevention of phagocytosis, evasion of host immune response, or even bacterial intracellularization. This variation in consequences for the host can largely be attributed to the many unique effector proteins between species (more than 100 have been identified), however, the proteins components of the type III secretion apparatus (T3SA) used to transfer these effectors are both structurally and functionally conserved. As there are still gaps in the current knowledge of how some of these T3SS proteins interact to regulate T3SA assembly and effector secretion, both structural and functional studies of these proteins are essential. In the work presented in this thesis, NMR studies and biophysical methods were used to characterize the interactions of T3SS tip proteins of S. typhimurium and P. aeruginosa with previously identified binding partners implicated in secretion control

Wildfire risk along the wildland-urban interface in Oklahoma in relation to encroaching eastern redcedar

Across the United States, the number of wildfires has been increasing, but this can especially be felt in the Great Plains, where some of the most drastic increases in wildfire frequency and size have occurred. While frequent fires should seem somewhat normal in a grassland biome like Oklahoma, after European settlement, fire suppression has allowed woody vegetation, like Eastern Redcedar, to encroach onto the grasslands and dominate native vegetation. This leads to state transitions of a grassland biome to a woodier ecosystem, which can cause more intense wildfires. The metropolitan areas outside of Oklahoma City and Tulsa have the highest housing and population densities at risk and largest magnitude of wildland-urban interface. But some rural areas like Hinton and Woodward to Watonga show a high risk to wildfire in proportion to their low populations. Landowners need to properly manage these areas to prevent fires rather than suppressing them

Morbidität und Mortalität der im Perinatalzentrum Dortmund behandelten Frühgeborenen mit einem Geburtsgewicht <1.500 g der Jahre 2003 und 2004

Ziel war es, die Qualität der Behandlung Frühgeborener im Perinatalzentrum Dortmund zu untersuchen, sie mit den Ergebnissen anderer nationaler und internationaler Studien zu vergleichen.Die Befunde von Frühgeborenen mit einem Geburtsgewicht von <1.500 g der Jahrgängen 2003/2004 wurden retrospektiv analysiert. Die Morbidität wurde zunächst quantitativ bestimmt durch Infektionen und Atemstörungen; qualitativ durch Herzvitien und schwere Verläufe einer NEC. Langfristig wurde das Outcome durch Gedeihprobleme und schwere intracerebrale Veränderungen bzw. Wahrnehmungsstörungen bedroht. Die Mortalität war im Wesentlichen bedingt durch die extreme Frühgeburtlichkeit, das sehr geringe Geburtsgewicht und angeborene Fehlbildungen. Morbidität und Mortalität entsprechen im Wesentlichen den Daten anderer großer Perinatalzentren. Ein hohes Niveau in der Versorgung (auch Reduktion von Frühgeburtlichkeit) kann nur über die effektive Zusammenarbeit mit den Geburtshelfern erzielt werden

NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts

Salmonella and Shigella bacteria require the type III secretion system (T3SS) to inject virulence proteins into their hosts and initiate infections. The tip proteins SipD and IpaD are critical components of the Salmonella and Shigella T3SS, respectively. Recently, SipD and IpaD have been shown to interact with bile salts, which are enriched in the intestines, and are hypothesized to act as environmental sensors for these enteric pathogens. Bile salts activate the Shigella T3SS but repress the Salmonella T3SS, and the mechanism of this differing response to bile salts is poorly understood. Further, how SipD binds to bile salts is currently unknown. Computer modeling predicted that IpaD binds the bile salt deoxycholate in a cleft formed by the N-terminal domain and the long central coiled coil of IpaD. Here, we used NMR methods to determine which SipD residues are affected by the interaction with the bile salts deoxycholate, chenodeoxycholate and taurodeoxcholate. The bile salts perturbed nearly the same set of SipD residues, however, the largest chemical shift perturbations occurred away from what was predicted for the bile salt binding site in IpaD. Our NMR results indicate that that bile salt interaction of SipD will be different from what was predicted for IpaD, suggesting a possible mechanism for the differing response of Salmonella and Shigella to bile salts

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available(1). Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment(2). Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice(3). Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown(4), employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)(5) and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate(5,6). Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants. Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for (15)N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein(7) (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state(8). We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE1(9)

NMR characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa

Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS) to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologs are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by CD and NMR spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residues 16–41 and 55–76. NMR backbone dynamics data show that the helices in PcrG have semi-rigid flexibility and they tumble as a single entity with similar backbone dynamics. NMR titrations show that the entire length of PcrG residues from 9–76 is involved in binding to PcrV. Thus the PcrG family of T3SS chaperone proteins is essentially partially folded

P1–058: The Hsp90 co‐chaperone FKBP51 produces neurotoxic tau oligomers: Implication for aging and Alzheimer’s disease

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152589/1/alzjjalz201305279.pd

Imbalance of Hsp70 family variants fosters tau accumulation

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154405/1/fsb2027004018.pd

Structure and function of PspA and Vipp1 N-terminal peptides: Insights into the membrane stress sensing and mitigation

The phage shock protein (Psp) response maintains integrity of the inner membrane (IM) in response to extracytoplasmic stress conditions and is widely distributed amongst enterobacteria. Its central component PspA, a member of the IM30 peripheral membrane protein family, acts as a major effector of the system through its direct association with the IM. Under non-stress conditions PspA also negatively regulates its own expression via direct interaction with the AAA + ATPase PspF. PspA has a counterpart in cyanobacteria called Vipp1, which is implicated in protection of the thylakoid membranes. PspA's and Vipp1's conserved N-terminal regions contain a putative amphipathic helix a (AHa) required for membrane binding. An adjacent amphipathic helix b (AHb) in PspA is required for imposing negative control upon PspF. Here, purified peptides derived from the putative AH regions of PspA and Vipp1 were used to directly probe their effector and regulatory functions. We observed direct membrane-binding of AHa derived peptides and an accompanying change in secondary structure from unstructured to alpha-helical establishing them as bona fide membrane-sensing AH's. The peptide-binding specificities and their effects on membrane stability depend on membrane anionic lipid content and stored curvature elastic stress, in agreement with full length PspA and Vipp1 protein functionalities. AHb of PspA inhibited the ATPase activity of PspF demonstrating its direct regulatory role. These findings provide new insight into the membrane binding and function of PspA and Vipp1 and establish that synthetic peptides can be used to probe the structure-function of the IM30 protein family

Epitope analysis following active immunization with tau proteins reveals immunogens implicated in tau pathogenesis

Abstract Background Abnormal tau hyperphosphorylation and its accumulation into intra-neuronal neurofibrillary tangles are linked to neurodegeneration in Alzheimer’s disease and similar tauopathies. One strategy to reduce accumulation is through immunization, but the most immunogenic tau epitopes have so far remained unknown. To fill this gap, we immunized mice with recombinant tau to build a map of the most immunogenic tau epitopes. Methods Non-transgenic and rTg4510 tau transgenic mice aged 5 months were immunized with either human wild-type tau (Wt, 4R0N) or P301L tau (4R0N). Each protein was formulated in Quil A adjuvant. Sera and splenocytes of vaccinated mice were collected to assess the humoral and cellular immune responses to tau. We employed a peptide array assay to identify the most effective epitopes. Brain histology was utilized to measure the effects of vaccination on tau pathology and inflammation. Results Humoral immune responses following immunization demonstrated robust antibody titers (up to 1:80,000 endpoint titers) to each tau species in both mice models. The number of IFN-γ producing T cells and their proliferation were also increased in splenocytes from immunized mice, indicating an increased cellular immune response, and tau levels and neuroinflammation were both reduced. We identified five immunogenic motifs within either the N-terminal (9-15 and 21-27 amino acids), proline rich (168-174 and 220-228 amino acids), or the C-terminal regions (427-438 amino acids) of the wild-type and P301L tau protein sequence. Conclusions Our study identifies five previously unknown immunogenic motifs of wild-type and mutated (P301L) tau protein. Immunization with both proteins resulted in reduced tau pathology and neuroinflammation in a tau transgenic model, supporting the efficacy of tau immunotherapy in tauopathy.http://deepblue.lib.umich.edu/bitstream/2027.42/109522/1/12974_2014_Article_152.pd