NMR studies of molecular interactions involved in the type III secretion system, SUMOylation, and the RNA binding protein HuR

Proteins are one of the most intriguing, versatile, and complex macromolecules in living systems. Proteins rarely function independently and perform their activities through a multitude of interactions with other proteins or molecules. Such molecular interactions are fundamental to almost all biological processes and their disruption is often associated with cellular irregularities and disease states. It is therefore of immense importance and interest to identify and characterize the binding interfaces of these biologically relevant molecular interactions. NMR spectroscopy is unparalleled in its ability to monitor molecular interactions in solution at atomic level over a wide range of affinities. In this body of work, various NMR methods were successfully used to study and characterize the protein-ligand interactions of three discrete systems: the bacterial type III secretion system (T3SS), the post-translational SUMO modification system, and post-transcriptional regulation by the RNA binding protein HuR. The T3SS is a macromolecular structure assembled by many Gram-negative bacterial pathogens, such as, Shigella flexneri, Yersinia pestis, and multi-drug resistant Pseudomonas aeruginosa, to cause infectious diseases. The structural component of the T3SS, the needle apparatus, consists of a base, a needle, a tip complex, and a translocon. Because the needle apparatus is exposed on the bacterial surface, present only among the pathogens, and essential for the virulence, disrupting the needle assembly is an attractive strategy for the development of novel anti-virulence drugs. However, this approach demands a detailed understanding of the protein-protein interactions involved in the needle assembly. Here, NMR methods were used to characterize the protein-protein interactions that are important in the assembly of the tip-translocon complex in the Shigella T3SS. Additionally, fragment-based screening was performed to identify small molecule binders of the tip proteins from Yersinia and Pseudomonas T3SS. The hits were subsequently validated and characterized using NMR spectroscopy. Our results provide novel insight into the assembly of the needle apparatus and reveal the first small molecules that directly bind to the tip proteins of Yersinia and Pseudomonas T3SS. Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process that can modulate biochemical and cell biological functions of the target protein substrate. SUMO E3 ligases are the enzymes that carry out the final step in SUMO conjugation pathway and facilitate the transfer of SUMO to the target protein. Prior to this work, SUMO binding by the PIAS family of E3 ligases was poorly understood. Here, using NMR spectroscopy, the protein-protein interactions involved in the SUMO-PIASy binding were characterized. The NMR binding studies surprisingly uncovered a novel SUMO-interacting motif in the E3 ligase PIASy, which was found to be essential for the ligase activity of PIASy. The RNA binding protein HuR binds to adenine- and uridine-rich elements (AREs) located in the untranslated region of target mRNAs, regulating their stability and translation. HuR-ARE interaction contributes to carcinogenesis by stabilization of oncogenic mRNAs. HuR is overexpressed in a broad range of human cancers and associated with poor clinical outcome. In vitro and in vivo studies have demonstrated that HuR is an attractive therapeutic target. Drugs that disrupt HuR-ARE interaction could potentially inhibit cancer growth and persistence. Here, a fungal natural product azaphilone (AZA-9) was identified as a novel disruptor of HuR-ARE interaction using fluorescence polarization based screening. AZA-9 binding to HuR was validated and characterized by NMR methods. Results of NMR studies suggest that AZA-9 binds in the ARE-binding cleft of HuR, and thus competitively inhibits the HuR-ARE interaction. The work presented in this dissertation illustrates the strength of NMR spectroscopy and its wide applicability as a tool to characterize and understand diverse interactions of proteins

STUDY OF SYNERGISTIC EFFECTS ON ANTIOXIDANT ACTIVITY AND ANTIMICROBIAL ACTIVITY OF POLYHERBAL FORMULATIONS CONTAINING FICUS SPECIES

Objective: The present study aims at screening the synergistic effect on the therapeutic efficiency of traditional herbal medicine Nalpamaradi Choorna and Nalpamaradi Keram, containing four Ficus species. The efficiency of formulations prepared by mixing crude drug is tested concerning their Antioxidant and Antibacterial activities. It will also provide and validate the use of these drugs in the current trend of targeted Combination Therapy for various neurodegenerative diseases.Methods: The in-vitro studies of the methanol extracts of the barks of the four individual plants, their different combinations, and the choorna were conducted by DPPH method, and the obtained EC50 values were compared to evaluate the synergistic effect. The antibacterial activity of Nalpamaradi Keram and the four Ficus plants was tested against two microorganisms Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) using agar well method.Results: The formulation prepared by mixing equal proportions of the four plants exhibited maximum %AA value of 93.36 % and EC50 value 8.00 µg/ml while the marketed drug showed maximum % AA of 87.30 % and an EC50 value of 29.0µg/ml. The combination of Ficus bengalensis (F. ben), Ficus racemosa (F. rac) and Ficus religiosa (F. rel) demonstrated the maximum % AA of 92.35 % and a very low EC50 value of 6.00µg/ml. All the samples except Ficus microcarpa (F. mic) exhibited antibacterial property against both the bacteria. Nalpamaradi Keram has shown the zone of inhibition of 20.0 mm against S. aureus and 18.0 mm against E. coli. Conclusion: The present investigation justifies the traditional use of these medicinal plants as antibacterial and antioxidant agents and validates their synergistic effect with improved activity in the formulations. Therefore, it is judicious to mix all the four Ficus species in the formulation of Nalpamaradi choorna and Nalpamaradi keram.Keywords: Antioxidant and synergistic activity, Antimicrobial activity, Ficus species, Polyherbal formulations, Radio protectivit

Rapid fermentation optimization for vaccine development

Pichia pastoris is widely used to produce heterologous proteins including vaccines such as hepatitis B [1]. P. pastoris is capable of achieve high cell density during fermentation and has the ability to secrete recombinant proteins. Fermentation optimization can be a time consuming and laborious process that can cause delays in early vaccine development. To overcome this, we have made use of parallel automated scalable fermentation technology. Four single-use parallel small-scale fermenters (ambr250 modular) were used to rapidly screen and optimize fermentation parameters. A non-replicative rotavirus (NRRV, PATH) vaccine was expressed in P. pastoris. The methylotrophic yeast was grown in fed-bath mode using chemically defined media with glycerol as carbon source and induced with methanol. Intact mass spectrometry was used to determine the distribution of full length and truncated species at different induction times during fermentation. Zymogram analysis was used to detect potential proteases present in the fermentation supernatant. The bioreactor operating parameters (pH, temperature, induction time and media composition) were optimized to improve cell growth and product yield. Resulting in product yields of ~ 1 g/L and purity levels of ~ 80% in the fermentation supernatant. Conditions were selected to reduce levels of truncation and increase production of full-length product. It was observed that temperature had little or no effect on mitigating product truncation; while fermentation pH and induction time had a greater effect, significantly reducing product truncation. In addition, zymogram analysis showed that levels of contaminating proteases in the supernatant were also affected by fermentation parameters and induction time. Cation exchange chromatography (CIEX) was used to purify the product directly from the fermentation supernatant showing that it is possible to integrate the up-stream process of fermentation with down-stream purification into a single procedure. Single-use small-scale fermenters are useful for a rapid screening and optimization of fermentation parameters for vaccine development. These results show that the bioreactor operating parameters have a great effect on both product yield and quality and fermentation parameters can be optimized to reduce degradation of secreted products. Reference 1. Hardy, E., et al., Large-scale production of recombinant hepatitis B surface antigen from Pichia pastoris. J Biotechnol, 2000. 77(2-3): p. 157-67

Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells.

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth

Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity

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

Structure and Biophysics of Type III Secretion in Bacteria

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus

NMR Identification of the Binding Surfaces Involved in the Salmonella and Shigella Type III Secretion Tip-Translocon Protein-Protein Interactions

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein–protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein–protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097–1107. © 2016 Wiley Periodicals, Inc