Synthesis and Stabilization of Highly Reactive Haloacetylium Ions.

The reactions of selected haloacetyl fluorides with the strong Lewis acids AsF5 and SbF5 were investigated in the aprotic solvent SO2ClF. Depending on the stoichiometric ratio of AsF5 or SbF5 and the haloacetyl fluorides, either O-coordinated adducts or the respective haloacetylium ions were isolated as solids. The compounds were characterized by low-temperature vibrational spectroscopy and single-crystal X-ray diffraction. [CClH2CO][Sb2F11] and [CH2FCO][Sb2F11] crystallize in the monoclinic space group P21 with two formula units per unit cell. The reactivity of the haloacetyl fluorides under Lewis acidic conditions is discussed based on quantum chemical calculations of fluoride ion affinities on the BP/def2SVPP level of theory. The haloacetylium ions are stabilized in the solid-state phase by intermolecular C···F contacts and electron donation of the fluorine ligands of the anions as well as intramolecular hyperconjugation.

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks.

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.

Automated solubility screening platform using computer vision.

Solubility screening is an essential, routine process that is often labor intensive. Robotic platforms have been developed to automate some aspects of the manual labor involved. However, many of the existing systems rely on traditional analytic techniques such as high-performance liquid chromatography, which require pre-calibration for each compound and can be resource consuming. In addition, automation is not typically end-to-end, requiring user intervention to move vials, establish analytical methods for each compound and interpret the raw data. We developed a closed-loop, flexible robotic system with integrated solid and liquid dosing capabilities that relies on computer vision and iterative feedback to successfully measure caffeine solubility in multiple solvents. After initial researcher input (<2 min), the system ran autonomously, screening five different solvent systems (20-80 min each). The resulting solubility values matched those obtained using traditional manual techniques.

CD20 as a gatekeeper of the resting state of human B cells.

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.

Organic synthesis in a modular robotic system driven by a chemical programming language.

The synthesis of complex organic compounds is largely a manual process that is often incompletely documented. To address these shortcomings, we developed an abstraction that maps commonly reported methodological instructions into discrete steps amenable to automation. These unit operations were implemented in a modular robotic platform by using a chemical programming language that formalizes and controls the assembly of the molecules. We validated the concept by directing the automated system to synthesize three pharmaceutical compounds, diphenhydramine hydrochloride, rufinamide, and sildenafil, without any human intervention. Yields and purities of products and intermediates were comparable to or better than those achieved manually. The syntheses are captured as digital code that can be published, versioned, and transferred flexibly between platforms with no modification, thereby greatly enhancing reproducibility and reliable access to complex molecules.

Exploring the applicability of density functional tight binding to transition metal ions. Parameterization for nickel with the spin-polarized DFTB3 model.

In this work, we explore the applicability and limitations of the current third order density functional tight binding (DFTB3) formalism for treating transition metal ions using nickel as an example. To be consistent with recent parameterization of DFTB3 for copper, the parametrization for nickel is conducted in a spin-polarized formulation and with orbital-resolved Hubbard parameters and their charge derivatives. The performance of the current parameter set is evaluated based on structural and energetic properties of a set of nickel-containing compounds that involve biologically relevant ligands. Qualitatively similar to findings in previous studies of copper complexes, the DFTB3 results are more reliable for nickel complexes with neutral ligands than for charged ligands; nevertheless, encouraging agreement is noted in comparison to the reference method, B3LYP/aug-cc-pVTZ, especially for structural properties, including cases that exhibit Jahn-Teller distortions; the structures also compare favorably to available X-ray data in the Cambridge Crystallographic Database for a number of nickel-containing compounds. As to limitations, we find it is necessary to use different d shell Hubbard charge derivatives for Ni(I) and Ni(II), due to the distinct electronic configurations for the nickel ion in the respective complexes, and substantial errors are observed for ligand binding energies, especially for charged ligands, d orbital splitting energies and splitting between singlet and triplet spin states for Ni(II) compounds. These observations highlight that future improvement in intra-d correlation and ligand polarization is required to enable the application of the DFTB3 model to complex transition metal ions. © 2018 Wiley Periodicals, Inc.

Progress in eradication of HCV in HIV positive patients with significant liver fibrosis in Vienna.

AIM: We aimed to investigate the efficacy of interferon and ribavirin-free sofosbuvir/ledipasvir (SOF/LDV) and ritonavir boosted paritaprevir/ombitasvir with or without dasabuvir (2D/3D) regimens in a real-life cohort of human immunodeficiency virus/hepatitis C virus (HIV/HCV) coinfected patients. The study focused on efficacy, need for changes in antiretroviral therapy (ART) due to drug-drug interaction (DDI), and treatment-associated changes in liver stiffness. METHODS: In this study 36 patients (n = 21 SOF/LDV and n = 15 2D/3D) were retrospectively analyzed. Depending on the genotype the following treatment regimens were used: HCV genotype (GT)-1: either SOF/LDV or 3D, no patient with HCV-GT2 was included, HCV-GT3: SOF/LDV, HCV-GT4: 2D. RESULTS: Approximately one third (35.3%) of patients were treatment-experienced and 13.9% had cirrhosis. Antiretroviral therapy had to be changed in 38.1% of SOF/LDV and 60% of 2D/3D patients prior to anti-HCV treatment due to expected DDIs. We observed sustained virologic response (SVR) rates of 100% in patients treated with SOF/LDV (19/19) and 2D/3D (14/14). One 2D/3D patient was lost to follow-up, while two SOF/LDV patients died during therapy from non-treatment-related causes. They were excluded from the analysis. Between baseline and follow-up liver stiffness decreased from 11.4 to 8.3 kPa (p = 0.008) and from 8.1 to 5.7 kPa (p = 0.001) in SOF/LDV and 2D/3D patients, respectively. CONCLUSIONS: We confirmed the excellent HCV eradication rates >95% in a real-life cohort of HIV/HCV coinfected patients treated with SOF/LDV and 2D/3D. We observed no HCV relapse or breakthrough. More patients treated with 2D/3D required a change in ART than patients treated with SOF/LDV. Additionally, HCV eradication led to a rapid decline in liver stiffness.

Interferon-free regimens improve health-related quality of life and fatigue in HIV/HCV-coinfected patients with advanced liver disease: A retrospective study.

Health-related quality of life (HRQoL) is impaired in HIV/HCV-coinfected patients (HIV/HCV) and further decreased by interferon (IFN)-based therapies. We aimed to investigate the impact of IFN- and ribavirin (RBV)-free therapies on HRQoL and fatigue.Thirty-three HIV/HCV-coinfected patients who underwent HCV therapy with sofosbuvir in combination with daclatasvir or ledipasvir were retrospectively studied and compared to 17 patients who received boceprevir (BOC)/PEGIFN/RBV. HRQoL (mental [MCS] and physical [PCS] component score) and fatigue were assessed using the SF-36 (Short Form 36 Health Survey) and the FSS (Fatigue Severity Scale), respectively. HRQoL/fatigue was evaluated at baseline (BL), midway, and 12 weeks after the end of treatment (FU).At BL, both domains of HRQoL as well as the severity of fatigue were significantly impaired in HIV/HCV, when compared to a healthy population. Already during treatment, IFN/RBV-free therapy improved physical health (PCS: 41.4 ±â€Š9.7 vs. 47.0 ±â€Š11.2; P < 0.01) and reduced fatigue (37.8 ±â€Š14.0 vs. 31.9 ±â€Š15.2; P = 0.01), whereas we observed a substantial worsening of both factors in patients treated with BOC/PEGIFN/RBV. Since these improvements were maintained, patients treated with IFN/RBV-free therapy reported an improvement in physical health (PCS: 41.4 ±â€Š9.7 vs. 45.8 ±â€Š12.7; P < 0.01) and fatigue (37.8 ±â€Š14.0 vs. 30.9 ±â€Š14.8; P = 0.04) at FU. While AIDS-patients had a higher severity of fatigue at BL and showed a reduction of fatigue (42.5 ±â€Š14.0 vs. 31.6 ±â€Š15.7; P = 0.01), mental health only improved in patients without AIDS (MCS: 35.7 ±â€Š5.3 vs.40.7 ±â€Š6.4; P = 0.04). HIV/HCV with severe fatigue at BL (>median BL-FSS) showed most pronounced improvements in severity of fatigue (49.7 ±â€Š7.0 vs. 32.0 ±â€Š16.7; P < 0.01).In contrast to IFN-based regimens, highly effective and well-tolerated IFN-/RBV-free regimens improve HRQoL (especially physical health) and fatigue already during treatment. All patients with HIV/HCV coinfection should be considered for HCV treatment; however, patients with severe fatigue should be prioritized.

Interferon-free treatment with sofosbuvir/daclatasvir achieves sustained virologic response in 100% of HIV/hepatitis C virus-coinfected patients with advanced liver disease.

AIM: We aimed to investigate the safety and efficacy of interferon (IFN) and ribavirin (RBV)-free therapy with sofosbuvir along with daclatasvir (SOF/DCV) in HIV/hepatitis C virus (HCV)-coinfected patients (HIV/HCV), who have an urgent need for effective antiviral therapy. We also assessed its impact on liver stiffness and liver enzymes. DESIGN: Thirty-one patients thoroughly documented HIV/HCV with advanced liver disease (advanced liver fibrosis and/or portal hypertension) who were treated with SOF/DCV were retrospectively studied. METHODS: The following treatment durations were applied: HCV-genotype (HCV-GT)1/4 without cirrhosis: 12 weeks; HCV-GT1/4 with cirrhosis: 24 weeks; HCV-GT3: 24 weeks; if HCV-RNA was detectable 4 weeks before the end of treatment, treatment was extended by 4 weeks at a time. RESULTS: Fifty-two percent of patients were treatment-experienced. The majority of patients had HCV-GT1 (68%), whereas HCV-GT3 and HCV-GT4 were observed in 23 and 10% of patients, respectively. Ninety-four percent had liver stiffness greater than 9.5 kPa or METAVIR fibrosis stage higher than F2 and 45% had liver stiffness above 12.5 kPa or METAVIR F4. Portal hypertension (HVPG ≥6 mmHg) and clinically significant portal hypertension (HVPG ≥10 mmHg) were observed in 67% (18/27) and 26% (7/27) of patients, respectively. Sustained virologic response 12 weeks after the end of treatment (SVR12) was achieved in 100% (31/31). Treatment with SOF/DCV was generally well tolerated and there were no treatment discontinuations. HCV eradication improved liver stiffness from 11.8 [interquartile range (IQR): 11.5 kPa] to 6.9 (IQR: 8.2) kPa [median change: -3.6 (IQR:5.2) kPa; P < 0.001] and decreased liver enzymes. The mean time period between treatment initiation and follow-up liver stiffness measurement was 32.7 ±â€Š1.2 weeks. CONCLUSION: IFN- and RBV-free treatment with SOF/DCV was well tolerated and achieved SVR12 in all HIV/HCV with advanced liver disease. It also significantly improved liver stiffness, suggesting anti-fibrotic and anti-portal hypertensive effects.

Advances in the management of HIV/HCV coinfection.

HCV coinfection has emerged as a major cause of non-AIDS-related morbidity and mortality in HIV-positive patients. As a consequence of the availability of modern combined antiretroviral therapy regimens, for optimally managed HIV/HCV-coinfected patients, the rates of liver fibrosis progression and the risk of liver-related events are increasingly similar to those of HCV-monoinfected patients. Moreover, our understanding of modulators of liver disease progression has greatly improved. In addition to immune status, endocrine, metabolic, genetic and viral factors are closely interrelated and might be important determinants of liver disease progression. In the last decade, a variety of serologic and radiographic tests for noninvasive liver disease staging have been extensively validated and are commonly used in HIV/HCV-coinfected patients. Sustained virologic response prevents end-stage liver disease, hepatocellular carcinoma, and death, with an even greater effect size in HIV-positive compared to HIV-negative patients. As interferon-free regimens achieve comparable rates of sustained virologic response in HIV-negative and HIV-positive patients, HIV/HCV-coinfected patients should from now on be referred to as a special, rather than a difficult-to-treat, population. Our comprehensive review covers all relevant aspects of HIV/HCV coinfection. Beginning with the changing epidemiology, it also provides new insights into the natural history of this condition and gives an overview on non-invasive techniques for the staging of liver disease. Furthermore, it outlines current recommendations for the treatment of acute hepatitis C and summarizes the unprecedented advances in the field of chronic hepatitis C therapy.

Treatment intensification with boceprevir in HIV-positive patients with acute HCV-genotype 1 infection at high risk for treatment failure.

BACKGROUND: According to current guidelines, the universal use of direct-acting antiviral agents in HIV-positive patients with acute hepatitis C (AHC) is not recommended. We aimed to evaluate the concept of treatment intensification with boceprevir (BOC) in HIV-positive patients with HCV-genotype 1 AHC (HIV/AHC-GT1) at high risk for failure to pegylated interferon/ribavirin therapy (PEGIFN/RBV). METHODS: Nineteen consecutive HIV-positive patients with HIV/AHC-GT1 who underwent antiviral therapy were studied retrospectively. Patients were treated with PEGIFN/RBV for 24 or 48 weeks, depending on rapid virologic response (RVR; undetectable HCV-RNA at treatment week [W] 4). Patients without complete early virologic response (cEVR; undetectable HCV-RNA at W 12) were offered treatment intensification with BOC at W 12, resulting in 36 weeks of BOC/PEGIFN/RBV triple therapy (total treatment duration: 48 weeks). RESULTS: Thirty-seven percent (7/19) of patients had an RVR and 74 % (14/19) of patients had a cEVR. BOC was used in four out of five patients who did not achieve cEVR and one patient elected to proceed with PEGIFN/RBV. Sustained virologic response (SVR; undetectable HCV-RNA 24 weeks after the end of treatment) rates were 100 % (14/14) among patients with cEVR treated with PEGIFN/RBV and 75 % (3/4) among patients without cEVR receiving BOC add-on. The patient without cEVR who preferred to continue with PEGIFN/RBV did not achieve SVR. Thus, the overall SVR rate was 89 % (17/19) in intention to treat analysis. CONCLUSIONS: BOC add-on in selected HIV/AHC-GT1 resulted in a high overall SVR rate. If 2nd generation direct-acting antiviral agents (DAAs) are not available, treatment intensification with BOC can be considered in HIV/AHC-GT1 at high risk for failure to PEGIFN/RBV.

Response-guided boceprevir-based triple therapy in HIV/HCV-coinfected patients: the HIVCOBOC-RGT study.

BACKGROUND: The HIVCOBOC-RGT study (NCT01925183) was the first study to evaluate response-guided shortening of the duration of boceprevir (BOC)-based triple therapy in human immunodeficiency virus (HIV)/hepatitis C virus genotype 1-coinfected patients (HIV/HCV-GT1). METHODS: After 4 weeks of pegylated interferon-α-2a/ribavirin (PEGIFN/RBV) lead-in, patients with target-not-detectable HCV-RNA at week 8 (rapid virologic response; LI4W-W8UTND) received 24 weeks of BOC/PEGIFN/RBV (total: 28 weeks [W28]). Patients with target-detectable HCV-RNA at week 8 received 44 weeks of BOC/PEGIFN/RBV (total: 48 weeks [W48]). RESULTS: Fourteen patients (67%) had LI4W-W8UTND and were eligible for the shortened W28 arm, while 7 (33%) patients were allocated to the W48 arm. No breakthrough or relapse occurred in the W28 arm, resulting in a sustained virologic response (SVR12TND) rate of 100% (12/12). In the W48 arm, the SVR12TND was 50% (3/6), with 3 patients meeting the futility rule at treatment week 12. The preliminary overall SVR12TND rate was 83% (15/18). Serious adverse events were observed in 5 (24%) patients, with 2 (10%) patients requiring surgical treatment of abscesses. CONCLUSIONS: The majority of HIV/HCV-GT1 were eligible for response-guided shortening of treatment duration to W28 and all of these patients had a SVR12TND. If second-generation direct-acting antivirals are not available, W28 of BOC-based triple therapy may be recommended.

Revisiting liver disease progression in HIV/HCV-coinfected patients: the influence of vitamin D, insulin resistance, immune status, IL28B and PNPLA3.

BACKGROUND & AIMS: To perform a comprehensive study on independent modulators of liver fibrosis progression and determinants of portal pressure considering immune status, insulin resistance (IR), serum 25-hydroxyvitamin D (25(OH)D) levels, genetic variants of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and interleukin 28B (IL28B) in a thoroughly documented cohort of HIV/hepatitis C-coinfected (HIV/HCV) patients. PATIENTS & METHODS: 25(OH)D deficiency (25(OH)DDEF), IR and low CD4(+) T-lymphocyte nadir (lowCD4NAD) were defined as 25(OH)D <20 ng × ml(-1) , HOMA-IR >2 and CD4nadir <200 cells × µl(-1) respectively. Liver fibrosis progression rate (FPR) was calculated as METAVIR F units divided by the number of years since HCV infection. Patients with a FPR > median FPR were assigned to the highFPR group. RESULTS: Among 86 HIV/HCV, the median FPR was 0.167 units × years(-1) . While the prevalence of prior alcohol abuse, lowCD4NAD and 25(OH)DDEF was higher among highFPR patients, the prevalence of IR was comparable. The association between 25(OH)DDEF and FPR was confirmed in a subgroup of patients with METAVIR stage F0/F1/F2 in which 25(OH)D levels are not affected by the severity of liver disease. The distribution of IL28B C/C and PNPLA3 non-C/C was similar, while PNPLA3 G/G was exclusively observed in highFPR patients. LowCD4NAD (OR: 2.95; 95% CI: 1.05-8.24; P = 0.039) and 25(OH)DDEF (OR: 5.62; 95% CI: 2.05-15.38; P = 0.001) were independently associated with highFPR and showed an additive effect. Portal pressure correlated with prior alcohol abuse, HCV-genotype 3, CD4(+) nadir and 25(OH)D levels. CONCLUSIONS: Two potentially modifiable factors, CD4(+) nadir and 25(OH)D levels, were both independent modulators of liver fibrosis progression and determinants of portal pressure. Further studies are warranted to assess the relevance of PNPLA3 for FPR in HIV/HCV.

A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons.

Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins.

Photosynthetic acclimation responses of maize seedlings grown under artificial laboratory light gradients mimicking natural canopy conditions.

In this study we assessed the ability of the C4 plant maize to perform long-term photosynthetic acclimation in an artificial light quality system previously used for analyzing short-term and long-term acclimation responses (LTR) in C3 plants. We aimed to test if this light system could be used as a tool for analyzing redox-regulated acclimation processes in maize seedlings. Photosynthetic parameters obtained from maize samples harvested in the field were used as control. The results indicated that field grown maize performed a pronounced LTR with significant differences between the top and the bottom levels of the plant stand corresponding to the strong light gradients occurring in it. We compared these data to results obtained from maize seedlings grown under artificial light sources preferentially exciting either photosystem II or photosystem I. In C3 plants, this light system induces redox signals within the photosynthetic electron transport chain which trigger state transitions and differential phosphorylation of LHCII (light harvesting complexes of photosystem II). The LTR to these redox signals induces changes in the accumulation of plastid psaA transcripts, in chlorophyll (Chl) fluorescence values F \rm s/F \rm m, in Chl a/b ratios and in transient starch accumulation in C3 plants. Maize seedlings grown in this light system exhibited a pronounced ability to perform both short-term and long-term acclimation at the level of psaA transcripts, Chl fluorescence values F \rm s/F \rm m and Chl a/b ratios. Interestingly, maize seedlings did not exhibit redox-controlled variations of starch accumulation probably because of its specific differences in energy metabolism. In summary, the artificial laboratory light system was found to be well-suited to mimic field light conditions and provides a physiological tool for studying the molecular regulation of the LTR of maize in more detail.

Photosystem II supercomplex remodeling serves as an entry mechanism for state transitions in Arabidopsis.

Within dense plant populations, strong light quality gradients cause unbalanced excitation of the two photosystems resulting in reduced photosynthetic efficiency. Plants redirect such imbalances by structural rearrangements of the photosynthetic apparatus via state transitions and photosystem stoichiometry adjustments. However, less is known about the function of photosystem II (PSII) supercomplexes in this context. Here, we show in Arabidopsis thaliana that PSII supercomplex remodeling precedes and facilitates state transitions. Intriguingly, the remodeling occurs in the short term, paralleling state transitions, but is also present in a state transition-deficient mutant, indicating that PSII supercomplex generation is independently regulated and does not require light-harvesting complex phosphorylation and movement. Instead, PSII supercomplex remodeling involves reversible phosphorylation of PSII core subunits (preferentially of CP43) and requires the luminal PSII subunit Psb27 for general formation and structural stabilization. Arabidopsis knockout mutants lacking Psb27 display highly accelerated state transitions, indicating that release of PSII supercomplexes is required for phosphorylation and subsequent movement of the antenna. Downregulation of PSII supercomplex number by physiological light treatments also results in acceleration of state transitions confirming the genetic analyses. Thus, supercomplex remodeling is a prerequisite and an important kinetic determinant of state transitions.

Identification of essential subunits in the plastid-encoded RNA polymerase complex reveals building blocks for proper plastid development.

The major RNA polymerase activity in mature chloroplasts is a multisubunit, Escherichia coli-like protein complex called PEP (for plastid-encoded RNA polymerase). Its subunit structure has been extensively investigated by biochemical means. Beside the "prokaryotic" subunits encoded by the plastome-located RNA polymerase genes, a number of additional nucleus-encoded subunits of eukaryotic origin have been identified in the PEP complex. These subunits appear to provide additional functions and regulation modes necessary to adapt transcription to the varying functional situations in chloroplasts. However, despite the enormous progress in genomic data and mass spectrometry techniques, it is still under debate which of these subunits belong to the core complex of PEP and which ones represent rather transient or peripheral components. Here, we present a catalog of true PEP subunits that is based on comparative analyses from biochemical purifications, protein mass spectrometry, and phenotypic analyses. We regard reproducibly identified protein subunits of the basic PEP complex as essential when the corresponding knockout mutants reveal an albino or pale-green phenotype. Our study provides a clearly defined subunit catalog of the basic PEP complex, generating the basis for a better understanding of chloroplast transcription regulation. In addition, the data support a model that links PEP complex assembly and chloroplast buildup during early seedling development in vascular plants.

Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression.

Photosynthetic light quality acclimation in plants involves redox-controlled changes in plastid gene expression. To study proteins potentially involved in this regulation, we isolated low-abundant chloroplast nucleic acid-binding proteins from the crucifere mustard (Sinapis alba) and investigated if photosynthetic redox signals affect their composition and/or oligomeric structure. We purified chloroplasts from plants subjected to light quality shifts and applied organelle lysates to heparin-Sepharose chromatography followed by 2-D blue native PAGE. We studied accumulation and structure of oligomeric protein complexes and applied MS/MS to identify them. We found ten oligomeric protein complexes of higher order and eleven smaller protein complexes or spots including plastid-encoded RNA polymerase (PEP), plastid transcriptionally active chromosome proteins, RNA-binding proteins, ribosomal subunits and chaperones. A translation elongation factor was found to be the only protein displaying major differences in its amounts in response to the growth lights. Furthermore, we found a novel thioredoxin as a subunit of the PEP, a 2-Cys-peroxiredoxin complex and a (soluble) ferredoxin:NADP-oxido-reductase, which represent potential redox regulator of plastid gene expression. A T-DNA knock-out line of the thioredoxin from Arabidopsis exhibits a yellowish-pale phenotype, demonstrating that this novel PEP subunit is essential for proper plastid development.

The role of phosphorylation in redox regulation of photosynthesis genes psaA and psbA during photosynthetic acclimation of mustard.

The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.

Fluorescence-based electrophoretic mobility shift assay in the analysis of DNA-binding proteins.

Changes in gene expression mediated by DNA-binding protein factors are a crucial part of many signal transduction pathways. Generally, these regulatory proteins are low abundant and thus their purification and characterisation is labour- and time-intensive. Here we describe a workflow for purification, characterisation and identification of DNA-binding proteins. We show the use of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and describe its advantages for a rapid and convenient screening for regulatory cis-elements. This involves a crude enrichment of nucleic acid binding proteins by heparin-Sepharose chromatography and the characterisation of fractions using overlapping fluorescence-labelled DNA probes spanning the promoter region of interest. The determined protein-binding sites can then be used for sequence-specific DNA-affinity chromatography to purify specifically interacting proteins. Finally, the DNA-binding complexes can be characterised and identified using two-dimensional EMSA, UV-cross-linking and mass spectrometry.