Atherosclerotic cardiovascular disease landscape in Singapore.

Cardiovascular disease (CVD) is the leading cause of death worldwide, accounting for over one-third of all deaths in Singapore. An analysis of age-standardized mortality rates (ASMR) for CVD in Singapore revealed a deceleration in the initial rapid decline in ASMR. A decrease in smoking prevalence may have contributed to the initial rapid decline in ASMR. Furthermore, other major risk factors, such as diabetes mellitus, hypertension, elevated low-density lipoprotein levels, and obesity, are steadily rising. Singapore's CVD economic burden is estimated to be 8.1 billion USD (11.5 billion SGD). The burden of CVD can only be reduced using individual and population-based approaches. Prevention programs must also be developed based on an understanding of risk trends. Therefore, this article attempts to capture the burden of CVD, trends in risk factor control, preventive care, disparities, and current unmet needs, particularly in atherosclerotic cardiovascular disease management in Singapore.

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 µM) and achieves "artemisinin-like" kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

Readmissions, Death and Its Associated Predictors in Heart Failure With Preserved Versus Reduced Ejection Fraction.

Background Data on rehospitalizations for heart failure (HF) in Asia are scarce. We sought to determine the burden and predictors of HF (first and recurrent) rehospitalizations and all-cause mortality in patients with HF and preserved versus reduced ejection fraction (preserved EF, ≥50%; reduced EF, <40%), in the multinational ASIAN-HF (Asian Sudden Cardiac Death in Heart Failure) registry. Methods and Results Patients with symptomatic (stage C) chronic HF were followed up for death and recurrent HF hospitalizations for 1 year. Predictors of HF hospitalizations or all-cause mortality were examined with Cox regression for time to first event and other methods for recurrent events analyses. Among 1666 patients with HF with preserved EF (mean age, 68±12 years; 50% women), and 4479 with HF with reduced EF (mean age, 61±13 years; 22% women), there were 642 and 2302 readmissions, with 28% and 45% attributed to HF, respectively. The 1-year composite event rate for first HF hospitalization or all-cause death was 11% and 21%, and for total HF hospitalization and all-cause death was 17.7 and 38.7 per 100 patient-years in HF with preserved EF and HF with reduced EF, respectively. In HF with preserved EF, consistent independent predictors of these clinical end points included enrollment as an inpatient, Southeast Asian location, and comorbid chronic kidney disease or atrial fibrillation. The same variables were predictive of outcomes in HF with reduced EF except atrial fibrillation, and also included Northeast Asian location, older age, elevated heart rate, decreased systolic blood pressure, diabetes, smoking, and non-usage of beta blockers. Conclusions One-year HF rehospitalization and mortality rates were high among Asian patients with HF. Predictors of outcomes identified in this study could aid in risk stratification and timely interventions. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01633398.

α-Synuclein Strains: Does Amyloid Conformation Explain the Heterogeneity of Synucleinopathies?

Synucleinopathies are a heterogeneous group of neurodegenerative diseases with amyloid deposits that contain the α-synuclein (SNCA/α-Syn) protein as a common hallmark. It is astonishing that aggregates of a single protein are able to give rise to a whole range of different disease manifestations. The prion strain hypothesis offers a possible explanation for this conundrum. According to this hypothesis, a single protein sequence is able to misfold into distinct amyloid structures that can cause different pathologies. In fact, a growing body of evidence suggests that conformationally distinct α-Syn assemblies might be the causative agents behind different synucleinopathies. In this review, we provide an overview of research on the strain hypothesis as it applies to synucleinopathies and discuss the potential implications for diagnostic and therapeutic purposes.

NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

C. elegans Models to Study the Propagation of Prions and Prion-Like Proteins.

A hallmark common to many age-related neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), is that patients develop proteinaceous deposits in their central nervous system (CNS). The progressive spreading of these inclusions from initially affected sites to interconnected brain areas is reminiscent of the behavior of bona fide prions in transmissible spongiform encephalopathies (TSEs), hence the term prion-like proteins has been coined. Despite intensive research, the exact mechanisms that facilitate the spreading of protein aggregation between cells, and the associated loss of neurons, remain poorly understood. As population demographics in many countries continue to shift to higher life expectancy, the incidence of neurodegenerative diseases is also rising. This represents a major challenge for healthcare systems and patients' families, since patients require extensive support over several years and there is still no therapy to cure or stop these diseases. The model organism Caenorhabditis elegans offers unique opportunities to accelerate research and drug development due to its genetic amenability, its transparency, and the high degree of conservation of molecular pathways. Here, we will review how recent studies that utilize this soil dwelling nematode have proceeded to investigate the propagation and intercellular transmission of prions and prion-like proteins and discuss their relevance by comparing their findings to observations in other model systems and patients.

Role of Fluid Biomarkers and PET Imaging in Early Diagnosis and its Clinical Implication in the Management of Alzheimer's Disease.

Clinical diagnosis of Alzheimer's disease (AD) is based on symptoms; however, the challenge is to diagnose AD at the preclinical stage with the application of biomarkers and initiate early treatment (still not widely available). Currently, cerebrospinal fluid (CSF) amyloid-ß 42 (Aß42) and tau are used in the clinical diagnosis of AD; nevertheless, blood biomarkers (Aß42 and tau) are less predictive. Amyloid-positron emission tomography (PET) imaging is an advancement in technology that uses approved radioactive diagnostic agents (florbetapir, flutemetamol, or florbetaben) to estimate Aß neuritic plaque density in adults with cognitive impairment evaluated for AD and other causes of cognitive decline. There is no cure for AD to date-the disease progression cannot be stopped or reversed; approved pharmacological agents (donepezil, galantamine, and rivastigmine; memantine) provide symptomatic treatment. However, the disease-modifying therapies are promising; aducanumab and CAD106 are in phase III trials for the early stages of AD. In conclusion, core CSF biomarkers reflect pathophysiology of AD in the early and late stages; the application of approved radiotracers have potential in amyloid-PET brain imaging to detect early AD.

Reducing INS-IGF1 signaling protects against non-cell autonomous vesicle rupture caused by SNCA spreading.

Aging is associated with a gradual decline of cellular proteostasis, giving rise to devastating protein misfolding diseases, such as Alzheimer disease (AD) or Parkinson disease (PD). These diseases often exhibit a complex pathology involving non-cell autonomous proteotoxic effects, which are still poorly understood. Using Caenorhabditis elegans we investigated how local protein misfolding is affecting neighboring cells and tissues showing that misfolded PD-associated SNCA/α-synuclein is accumulating in highly dynamic endo-lysosomal vesicles. Irrespective of whether being expressed in muscle cells or dopaminergic neurons, accumulated proteins were transmitted into the hypodermis with increasing age, indicating that epithelial cells might play a role in remote degradation when the local endo-lysosomal degradation capacity is overloaded. Cell biological and genetic approaches revealed that inter-tissue dissemination of SNCA was regulated by endo- and exocytosis (neuron/muscle to hypodermis) and basement membrane remodeling (muscle to hypodermis). Transferred SNCA conformers were, however, inefficiently cleared and induced endo-lysosomal membrane permeabilization. Remarkably, reducing INS (insulin)-IGF1 (insulin-like growth factor 1) signaling provided protection by maintaining endo-lysosomal integrity. This study suggests that the degradation of lysosomal substrates is coordinated across different tissues in metazoan organisms. Because the chronic dissemination of poorly degradable disease proteins into neighboring tissues exerts a non-cell autonomous toxicity, this implies that restoring endo-lysosomal function not only in cells with pathological inclusions, but also in apparently unaffected cell types might help to halt disease progression.Abbreviations: AD: Alzheimer disease; BM: basement membrane; BWM: body wall muscle; CEP: cephalic sensilla; CLEM: correlative light and electron microscopy; CTNS-1: cystinosin (lysosomal protein) homolog; DA: dopaminergic; DAF-2: abnormal dauer formation; ECM: extracellular matrix; FLIM: fluorescence lifetime imaging microscopy; fps: frames per second; GFP: green fluorescent protein; HPF: high pressure freezing; IGF1: insulin-like growth factor 1; INS: insulin; KD: knockdown; LMP: lysosomal membrane permeabilization; MVB: multivesicular body; NOC: nocodazole; PD: Parkinson disease; RFP: red fluorescent protein; RNAi: RNA interference; sfGFP: superfolder GFP; SNCA: synuclein alpha; TEM: transmission electron microscopy; TNTs: tunneling nanotubes; TCSPC: time correlated single photon counting; YFP: yellow fluorescent protein.

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit.

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50<0.1µM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.

PI4 Kinase Is a Prophylactic but Not Radical Curative Target in Plasmodium vivax-Type Malaria Parasites.

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo.

Identification of Three Novel Ring Expansion Metabolites of KAE609, a New Spiroindolone Agent for the Treatment of Malaria, in Rats, Dogs, and Humans.

KAE609 [(1'R,3'S)-5,7'-dichloro-6'-fluoro-3'-methyl-2',3',4',9'-tetrahydrospiro[indoline-3,1'-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent being developed for the treatment of malaria. After oral dosing of KAE609 to rats and dogs, the major radioactive component in plasma was KAE609. An oxidative metabolite, M18, was the prominent metabolite in rat and dog plasma. KAE609 was well absorbed and extensively metabolized such that low levels of parent compound (≤11% of the dose) were detected in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways (≥93% of the dose) in the feces of rats and dogs. M37 and M23 were the major metabolites in rat and dog feces, respectively. Among the prominent metabolites of KAE609, the isobaric chemical species, M37, was observed, suggesting the involvement of an isomerization or rearrangement during biotransformation. Subsequent structural elucidation of M37 revealed that KAE609, a spiroindolone, undergoes an unusual C-C bond cleavage, followed by a 1,2-acyl shift to form a ring expansion metabolite M37. The in vitro metabolism of KAE609 in hepatocytes was investigated to understand this novel biotransformation. The metabolism of KAE609 was qualitatively similar across the species studied; thus, further investigation was conducted using human recombinant cytochrome P450 enzymes. The ring expansion reaction was found to be primarily catalyzed by cytochrome P450 (CYP) 3A4 yielding M37. M37 was subsequently oxidized to M18 by CYP3A4 and hydroxylated to M23 primarily by CYP1A2. Interestingly, M37 was colorless, whereas M18 and M23 showed orange yellow color. The source of the color of M18 and M23 was attributed to their extended conjugated system of double bonds in the structures.

The Pseudomonas aeruginosa Isohexenyl Glutaconyl Coenzyme A Hydratase (AtuE) Is Upregulated in Citronellate-Grown Cells and Belongs to the Crotonase Family.

Pseudomonas aeruginosa is one of only a few Pseudomonas species that are able to use acyclic monoterpenoids, such as citronellol and citronellate, as carbon and energy sources. This is achieved by the acyclic terpene utilization pathway (Atu), which includes at least six enzymes (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG) and is coupled to a functional leucine-isovalerate utilization (Liu) pathway. Here, quantitative proteome analysis was performed to elucidate the terpene metabolism of P. aeruginosa. The proteomics survey identified 187 proteins, including AtuA to AtuG and LiuA to LiuE, which were increased in abundance in the presence of citronellate. In particular, two hydratases, AtuE and the PA4330 gene product, out of more than a dozen predicted in the P. aeruginosa proteome showed an increased abundance in the presence of citronellate. AtuE (isohexenyl-glutaconyl coenzyme A [CoA] hydratase; EC 4.2.1.57) most likely catalyzes the hydration of the unsaturated distal double bond in the isohexenyl-glutaconyl-CoA thioester to yield 3-hydroxy-3-isohexenyl-glutaryl-CoA. Determination of the crystal structure of AtuE at a 2.13-Å resolution revealed a fold similar to that found in the hydratase (crotonase) superfamily and provided insights into the nature of the active site. The AtuE active-site architecture showed a significantly broader cavity than other crotonase superfamily members, in agreement with the need to accommodate the branched isoprenoid unit of terpenes. Glu139 was identified to be a potential catalytic residue, while the backbone NH groups of Gly116 and Gly68 likely form an oxyanion hole. The present work deepens the understanding of terpene metabolism in Pseudomonas and may serve as a basis to develop new strategies for the biotechnological production of terpenoids.

Analysis of the molecular response of Pseudomonas putida KT2440 to the next-generation biofuel n-butanol.

To increase the efficiency of biocatalysts a thorough understanding of the molecular response of the biocatalyst to precursors, products and environmental conditions applied in bioconversions is essential. Here we performed a comprehensive proteome and phospholipid analysis to characterize the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the next-generation biofuel n-butanol. Using complementary quantitative proteomics approaches we were able to identify and quantify 1467 proteins, corresponding to 28% of the total KT2440 proteome. 256 proteins were altered in abundance in response to n-butanol. The proteome response entailed an increased abundance of enzymes involved in n-butanol degradation including quinoprotein alcohol dehydrogenases, aldehyde dehydrogenases and enzymes of fatty acid beta oxidation. From these results we were able to construct a pathway for the metabolism of n-butanol in P. putida. The initial oxidation of n-butanol is catalyzed by at least two quinoprotein ethanol dehydrogenases (PedE and PedH). Growth of mutants lacking PedE and PedH on n-butanol was significantly impaired, but not completely inhibited, suggesting that additional alcohol dehydrogenases can at least partially complement their function in KT2440. Furthermore, phospholipid profiling revealed a significantly increased abundance of lyso-phospholipids in response to n-butanol, indicating a rearrangement of the lipid bilayer. BIOLOGICAL SIGNIFICANCE: n-butanol is an important bulk chemical and a promising alternative to gasoline as a transportation fuel. Due to environmental concerns as well as increasing energy prices there is a growing interest in sustainable and cost-effective biotechnological production processes for the production of bulk chemicals and transportation fuels from renewable resources. n-butanol fermentation is well established in Clostridiae, but the efficiency of n-butanol production is mainly limited by its toxicity. Therefore bacterial strains with higher intrinsic tolerance to n-butanol have to be selected as hosts for n-butanol production. Pseudomonas bacteria are metabolically very versatile and exhibit a high intrinsic tolerance to organic solvents making them suitable candidates for bioconversion processes. A prerequisite for a potential production of n-butanol in Pseudomonas bacteria is a thorough understanding of the molecular adaption processes caused by n-butanol and the identification of enzymes involved in n-butanol metabolization. This work describes the impact of n-butanol on the proteome and the phospholipid composition of the reference strain P. putida KT2440. The high proteome coverage of our proteomics survey allowed us to reconstruct the degradation pathway of n-butanol and to monitor the changes in the energy metabolism of KT2440 induced by n-butanol. Key enzymes involved in n-butanol degradation identified in study will be interesting targets for optimization of n-butanol production in Pseudomonads. The present work and the identification of key enzymes involved in butanol metabolism may serve as a fundament to develop new or improve existing strategies for the biotechnological production of the next-generation biofuel n-butanol in Pseudomonads.

Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin.

Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the ß-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. BIOLOGICAL SIGNIFICANCE: The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a proteome wide scale. The high proteome coverage of our proteomics survey allowed us to analyze the regulation of whole protein networks instead of single proteins. We were able to reconstruct the complete degradation pathway of vanillin and to monitor the changes in the energy metabolism of KT2440 induced by vanillin as sole carbon source. Vanillin dehydrogenase (Vdh) was not mandatory for vanillin degradation in KT2440 and may be substituted by other aldehyde dehydrogenases that were up-regulated in a wild-type as well as in a Vdh-deficient strain in the presence of vanillin. Aldehyde dehydrogenases, vanillin specific porins and efflux pump systems identified in study will be interesting targets for optimization of vanillin production in Pseudomonas bacteria. Furthermore, several mechanisms of solvent tolerance were induced by vanillin in KT2440. These include increased abundance of several efflux pump systems, chaperones as well as enzymes involved in cyclopropane fatty acid synthesis and trehalose formation. The present work will deepen the understanding of metabolism of aromatic compounds in P. putida and may lead to a more comprehensive understanding of solvent tolerance mechanisms in Gram-negative bacteria in general. Moreover, it will serve as a basis for further strain developments for a biotechnological production of vanillin in P. putida KT2440 or other Pseudomonas strains, highlighting the role of proteomics surveys as a powerful screening technology.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Ethylene glycol metabolism by Pseudomonas putida.

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane.

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.

Total synthesis confirms laetirobin as a formal Diels-Alder adduct.

Laetirobin, isolated from a parasitic fungus host-plant relationship, was synthesized in six practical steps with an overall yield of 12% from commercially available 2,4-dihydroxyacetophenone. Because the product is a pseudosymmetric tetramer of benzo[b]furans, each step of the synthesis was designed to involve tandem operations. Highlights include: 1) the double Sonogashira reaction of a bis(alkyne), 2) the practical copper(I)-mediated formation of a bis(benzo[b]furan), and 3) the biomimetic [4+2] dimerization and unexpected cationic [5+2] annulation of gem-diaryl alkene precursors. Preliminary structure-activity relationship data between the isomeric [4+2] and [5+2] tetramers revealed only the natural product to possess promising anticancer potential. Specifically, laetirobin is capable of blocking tumor cell division (mitosis) and invoking programmed cell death (apoptosis).

Laetirobin from the parasitic growth of Laetiporus sulphureus on Robinia pseudoacacia.

(+/-)-Laetirobin (1) was isolated as a cytostatic lead from Laetiporus sulphureus growing parasitically on the black locust tree, Robinia pseudoacacia, by virtue of a reverse-immunoaffinity system. Using an LC/MS procedure, milligram quantities of (+/-)-laetirobin (1) were obtained, and the structure of 1 was elucidated by X-ray crystallography and confirmed by NMR spectroscopy. Preliminary cellular studies indicated that (+/-)-laetirobin (1) rapidly enters in tumor cells, blocks cell division at a late stage of mitosis, and invokes apoptosis.

Characterization of platelet proteins using peptide centric proteomics.

In modern proteomics, undersampling of low abundant, cumbersome, and hydrophobic proteins states one of the major problems. To overcome this, especially in two 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) eminent drawbacks, the so-called peptide-centric techniques have been developed. These approaches do not separate proteins prior to digestion, but instead proteolytically generate peptide mixtures after it. However, by this procedure already complex protein mixtures become even more extensive peptide mixtures. Particularly, when dealing with large proteomes, the generated sample complexity is vast and therefore difficult to analyze. When separated and analyzed by LC/MS, too many peptides may enter the mass spectrometer at a certain time point, and only a small fraction of ions is selected for subsequent MS/MS analysis. Although protein hydrophobicity and size play minor roles (as long as protease cleavage sites are accessible), low copy number can severely limit identification rates. To reduce the amount of peptides entering the mass spectrometer simultaneously without the loss of overall proteomic information, different techniques have been developed. Among these, an approach is represented by COFRADIC (Combined Fractional Diagonal Chromatography). COFRADIC is a chromatography-based technique enabling the sorting of peptides due to retention time shifts after a specific modification step. In the original approach, a complex peptide mixture is separated by a primary RP-HPLC (reversed-phase high-performance liquid chromatography) run and fractions are retained. Subsequently, these fractions are modified to specifically change retention times of peptides and separated in one or more secondary RP-HPLC runs. In this chapter, COFRADIC approaches for methionine or cysteine containing as well as N-terminal peptides are described. Besides the reduction of sample complexity, the major advantage of COFRADIC might be seen in its versatility. Nearly every feature unique for a subset of peptides, which can be specifically modified by a sorting reaction, is accessible for COFRADIC. Among these are protein phosphorylation, N-glycosylation, and in vivo protein processing sites. Finally, COFRADIC allows the analysis of large numbers of samples and is highly automatable.