Deciphering the Mechanism of E. coli tat Protein Transport: Kinetic Substeps and Cargo Properties

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA. The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (∆psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ∆psi, the delay likely represents some preparatory step that results in a transport competent translocon. In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system’s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery’s ability to transport substrates determined by the protein’s molecular weight, as well as by its dimensions. In total, these results suggest a dynamic translocon that undergoes functionally significant, ∆psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape

Deciphering the Mechanism of E. coli tat Protein Transport: Kinetic Substeps and Cargo Properties

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA. The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (∆psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ∆psi, the delay likely represents some preparatory step that results in a transport competent translocon. In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system’s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery’s ability to transport substrates determined by the protein’s molecular weight, as well as by its dimensions. In total, these results suggest a dynamic translocon that undergoes functionally significant, ∆psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape

Impact of food, alcohol and pH on modified-release hydrocortisone developed to treat congenital adrenal hyperplasia.

BACKGROUND: We developed a modified-release hydrocortisone, Chronocort®, to replace the cortisol rhythm in patients with congenital adrenal hyperplasia. Food, alcohol and pH affect drug absorption and it is important to assess their impact when replicating a physiological rhythm. SUBJECTS AND METHODS: In vitro dissolution to study impact of alcohol and pH on Chronocort®. A Phase 1, three-period, cross over study in 18 volunteers to assess the impact of food on Chronocort® and to compare bioavailability to immediate-release hydrocortisone. RESULTS: In vitro dissolution of Chronocort® was not affected by gastrointestinal pH up to 6.0 nor by an alcohol content up to 20 % v/v. Food delayed and reduced the rate of absorption of Chronocort® as reflected by a longer Tmax (fed vs fasted: 6.75 hrs vs 4.5 hrs, p=0005) and lower Cmax (549.49 vs 708.46, nmol/L, ratio 77% with CI 71 - 85). Cortisol exposure was similar in fed and fasted state: Geo LSmean ratio (CI) AUC0 t for fed/fasted was 108.33% (102.30 - 114.72%). Cortisol exposure was higher for Chronocort® compared to immediate-release hydrocortisone: Geo LSmean ratios (CI) 118.83% (111.58 - 126.54%); however, derived free cortisol showed cortisol exposure CIs were within 80.0 125.0 %: Geo LSmean ratio (CI) for AUC0 t 112.73% (105.33 - 120.65%). CONCLUSIONS: Gastric pH ≤ 6.0 and alcohol do not effect hydrocortisone release from Chronocort®. Food delays Chronocort® absorption but cortisol exposure is similar in the fasted and fed state and exposure as assessed by free cortisol is similar between Chronocort® and immediate-release hydrocortisone

Impact of the COVID-19 global pandemic on symptomatic diagnosis of cancer - the view from primary care

The entire landscape of cancer management in primary care, from case identification to the management of those living with and beyond cancer, is evolving rapidly in the face of the coronavirus (COVID-19) pandemic.1 In a climate of fear and mandated avoidance of all but essential clinical services, delays in patient, population and healthcare system responses to suspected cancer symptoms seem inevitable

The Pfs230 N-terminal fragment, Pfs230D1+: expression and characterization of a potential malaria transmission-blocking vaccine candidate

This work is licensed under a Creative Commons Attribution 4.0 International License.Background Control and elimination of malaria can be accelerated by transmission-blocking interventions such as vaccines. A surface antigen of Plasmodium falciparum gametocytes, Pfs230, is a leading vaccine target antigen, and has recently progressed to experimental clinical trials. To support vaccine product development, an N-terminal Pfs230 antigen was designed to increase yield, as well as to improve antigen quality, integrity, and homogeneity. Methods A scalable baculovirus expression system was used to express the Pfs230D1+ construct (aa 552–731), which was subsequently purified and analysed. Pfs230D1+ was designed to avoid glycosylation and protease digestion, thereby potentially increasing homogeneity and stability. The resulting Pfs230D1+ protein was compared to a previous iteration of the Pfs230 N-terminal domain, Pfs230C1 (aa 443–731), through physiochemical characterization and in vivo analysis. The induction of functional antibody responses was confirmed via the standard membrane feeding assay (SMFA). Results Pfs230D1+ was produced and purified to an overall yield of 23 mg/L culture supernatant, a twofold yield increase over Pfs230C1. The Pfs230D1+ protein migrated as a single band via SDS-PAGE and was detected by anti-Pfs230C1 monoclonal antibodies. Evaluation by SDS-PAGE, chromatography (size-exclusion and reversed phase) and capillary isoelectric focusing demonstrated the molecule had improved homogeneity in terms of size, conformation, and charge. Intact mass spectrometry confirmed its molecular weight and that it was free of glycosylation, a key difference to the prior Pfs230C1 protein. The correct formation of the two intramolecular disulfide bonds was initially inferred by binding of a conformation specific monoclonal antibody and directly confirmed by LC/MS and peptide mapping. When injected into mice the Pfs230D1+ protein elicited antibodies that demonstrated transmission-reducing activity, via SMFA, comparable to Pfs230C1. Conclusion By elimination of an O-glycosylation site, a potential N-glycosylation site, and two proteolytic cleavage sites, an improved N-terminal Pfs230 fragment was produced, termed D1+, which is non-glycosylated, homogeneous, and biologically active. An intact protein at higher yield than that previously observed for the Pfs230C1 fragment was achieved. The results indicate that Pfs230D1+ protein produced in the baculovirus expression system is an attractive antigen for transmission-blocking vaccine development

Asporin is a stromally expressed marker associated with prostate cancer progression.

BACKGROUND: Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes. METHODS: We have used Kaplan-Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells. RESULTS: We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan-Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p53 inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines. CONCLUSIONS: Our results suggest that ASPN is a stromally expressed biomarker that correlates with disease progression, and is observed in reactive stroma. ASPN expression in stroma may be part of a stromal response to aggressive tumour subtypes.British Journal of Cancer advance online publication, 2 February 2017; doi:10.1038/bjc.2017.15 www.bjcancer.com

Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

Felisbino S. received a fellowship from the Sao Paulo Research Foundation (FAPESP) ref. 2013/08830-2 and 2013/06802-1. Anne Y Warren research time funded by: Cambridge Biomedical Research Centre.Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa.Publisher PDFPeer reviewe

Developability Assessment of Physicochemical Properties and Stability Profiles of HIV-1 BG505 SOSIP.664 and BG505 SOSIP.v4.1-GT1.1 gp140 Envelope Glycoprotein Trimers as Candidate Vaccine Antigens

This work is licensed under a Creative Commons Attribution 4.0 International License.The induction of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an effective vaccine against HIV-1. A soluble, trimeric, germline (gI) bNAb-targeting variant of the HIV-1 envelope glycoprotein (termed BG505 SOSIP.v4.1-GT1.1 gp140, abbreviated to GT1.1) has recently been developed. Here, we have compared this new immunogen with the parental trimer from which it was derived, BG505 SOSIP.664 gp140. We used a comprehensive suite of biochemical and biophysical methods to determine physicochemical similarities and differences between the 2 trimers, and thereby assessed whether additional formulation development efforts were needed for the GT1.1 vaccine candidate. The overall higher order structure and oligomeric states of the 2 vaccine antigens were quite similar, as were their thermal, chemical, and colloidal stability profiles, as evaluated during accelerated stability studies. Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. This developability assessment of the BG505 GT1.1 vaccine antigen supports using the formulation and storage conditions previously identified for the parental SOSIP.664 trimer and enables the development of GT1.1 for phase I clinical studies.Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Development (OPP1147661)Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Development (OPP1153692)NIH HIVRAD grant P01 AI 110657Aids Fonds grant 201601

High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors

The International Surface Pressure Databank version 2

The International Surface Pressure Databank (ISPD) is the world's largest collection of global surface and sea-level pressure observations. It was developed by extracting observations from established international archives, through international cooperation with data recovery facilitated by the Atmospheric Circulation Reconstructions over the Earth (ACRE) initiative, and directly by contributing universities, organizations, and countries. The dataset period is currently 1768–2012 and consists of three data components: observations from land stations, marine observing systems, and tropical cyclone best track pressure reports. Version 2 of the ISPD (ISPDv2) was created to be observational input for the Twentieth Century Reanalysis Project (20CR) and contains the quality control and assimilation feedback metadata from the 20CR. Since then, it has been used for various general climate and weather studies, and an updated version 3 (ISPDv3) has been used in the ERA-20C reanalysis in connection with the European Reanalysis of Global Climate Observations project (ERA-CLIM). The focus of this paper is on the ISPDv2 and the inclusion of the 20CR feedback metadata. The Research Data Archive at the National Center for Atmospheric Research provides data collection and access for the ISPDv2, and will provide access to future versions