The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR

The low-density lipoprotein receptor （LDLR） mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins （LDLs）. Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia （FH） is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafdomycin A1 or addition of the irreversible serine protease inhibitors, 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF） and 3,4-dichloroisocoumarin （DCI）, prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.     

Diversity and Versatility of the Thermotoga maritima Sugar Kinome

Sugar phosphorylation is an indispensable committed step in a large variety of sugar catabolic pathways, which are major suppliers of carbon and energy in heterotrophic species. Specialized sugar kinases that are indispensable for most of these pathways can be utilized as signature enzymes for the reconstruction of carbohydrate utilization machinery from microbial genomic and metagenomic data. Sugar kinases occur in several structurally distinct families with various partially overlapping as well as yet unknown substrate specificities that often cannot be accurately assigned by homology-based techniques. A subsystems-based metabolic reconstruction combined with the analysis of genome context and followed by experimental testing of predicted gene functions is a powerful approach of functional gene annotation. Here we applied this integrated approach for functional mapping of all sugar kinases constituting an extensive and diverse sugar kinome in the thermophilic bacterium Thermotoga maritima. Substrate preferences of 14 kinases mainly from the FGGY and PfkB families were inferred by bioinformatics analysis and biochemically characterized by screening with a panel of 45 different carbohydrates. Most of the analyzed enzymes displayed narrow substrate preferences corresponding to their predicted physiological roles in their respective catabolic pathways. The observed consistency supports the choice of kinases as signature enzymes for genomics-based identification and reconstruction of sugar utilization pathways. Use of the integrated genomic and experimental approach greatly speeds up the identification of the biochemical function of unknown proteins and improves the quality of reconstructed pathways.     

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.     

Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible Degrader of the LDLR (IDOL)

We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease.     

Versatility and reliability of combined flaps of the subscapular system

One-stage reconstructions of complex or unusually large defects frequently require composite tissue transfers. The various components of these "chimeric" flaps facilitate three-dimensional reconstructions or the coverage of large surface defects. Data from 36 combined flaps from the subscapular arterial system are demonstrated in this series. Defect locations were evenly distributed between the upper and lower extremities. Eighty-three percent were two-component flaps, and 17 percent contained three or more various tissue components. Overall flap survival was 97 percent. Major complications included vascular revisions in four patients and seven secondary skin transplantations. Five cases contained osseous components. The independent mobility of skin, muscle, and bone proved to be a major advantage in the reconstruction of compound defects. Donor-site morbidity was acceptable; the most frequent donor-site complication was persistent seroma in 9 of 36 patients (25 percent). Patient satisfaction was high. Ninety-one percent were satisfied with the operative result and would undergo the operation again. Eighty-six percent accepted the aesthetic appearance of the donor site. The data demonstrate that these complex flap procedures are extremely reliable and versatile, thus avoiding multiple reconstructive procedures and achieving excellent reconstructive results with acceptable donor-site morbidity.     

Determining the Advantages,Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST)

The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.     

Use of Antiviral T-Lymphocyte Clones to Characterize Antigen Presentation and T-Lymphocyte Subsets

The ability to isolate homogeneous populations of antiviral T lymphocytes from immune mice has led to insight into a variety of areas in cellular immunology. It has permitted the characterization of the distinct pathways of antigen processing and presentation to CD8⁺, Class I MHC-restricted and CD4⁺, Class II MHC-restricted cytolytic T lymphocytes as well as the identification of antigenic epitopes for T lymphocytes. In addition,in vivoeffector function of CD8⁺and CD4⁺cytolytic T-cell clones in protection from lethal viral pneumonia in a murine model of influenza virus infection has been demonstrated. Since the identification of CD4⁺T-lymphocyte helper subsets based on the lymphokine profiles of clonal populations, much interest has been focused on the role of specific cytokines in ultimately determining the effector functions of those cells. The protocol presented in this paper has been used to isolate Th1 and Th2 clones in a viral infectious disease model that has enabled us to further investigate the role of specific cytokines in controlling viral infection.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

A Kinetic Approach to Characterize the Electrostatic Environments of Thiol Groups in Proteins

In this study, we synthesized a zwitterionic DTNB derivative, 5-(2-aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios,k_ADNB/k_DTNB, varied from 0.22 for reactions with low-molecular-weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. Ak_ADNB/k_DTNBratio of 200 for Cys-34 of BSA appears to reflect a very anionic environment.k_ADNB/k_DTNBratios of 6 and 1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA.k_ADNB/k_DTNBratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins.     

A Liquid Chromatography-Mass Spectrometry-based Approach to Characterize the Substrate Specificity of Mammalian Heparanase

Extracellular heparanase activity releases growth factors and angiogenic factors from heparan sulfate (HS) storage sites and alters the integrity of the extracellular matrix. These activities lead to a loss of normal cell matrix adherent junctions and correlate with invasive cellular phenotypes. Elevated expression of heparanase is associated with several human cancers and with vascular remodeling. Heparanase cleaves only a limited fraction of glucuronidic linkages in HS. There have been few investigations of the functional consequences of heparanase activity, largely due to the heterogeneity and complexity of HS. Here, we report a liquid chromatography-mass spectrometry (LC-MS)-based approach to profile the terminal structures created by heparanase digestion and reconstruct the heparanase cleavage sites from the products. Using this method, we demonstrate that heparanase cleaves at the non-reducing side of highly sulfated HS domains, exposing cryptic growth factor binding sites. This cleavage pattern is observed in HS from several tissue sources, regardless of overall sulfation degree, indicating a common recognition pattern. We further demonstrate that heparanase cleavage of HS chains leads to increased ability to support FGF2-dependent cell proliferation. These results suggest a new mechanism to explain how heparanase might potentiate the uncontrolled cell proliferation associated with cancer through its ability to activate nascent growth factor-promoting domains within HS.     

Comparative Genomic and Phylogenetic Approaches to Characterize the Role of Genetic Recombination in Mycobacterial Evolution

The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer (M. abscessus to Segniliparus and M. smegmatis to Streptomyces), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case (M. vanbaalenii to Streptomyces) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is relatively sparse, the acquisition of genes from non-mycobacterial lineages is a significant feature of mycobacterial evolution.     

Using Health Care Utilization and Publication Patterns to Characterize the Research Portfolio and to Plan Future Research Investments

Objective

Government funders of biomedical research are under increasing pressure to demonstrate societal benefits of their investments. A number of published studies attempted to correlate research funding levels with the societal burden for various diseases, with mixed results. We examined whether research funded by the Department of Veterans Affairs (VA) is well aligned with current and projected veterans’ health needs. The organizational structure of the VA makes it a particularly suitable setting for examining these questions.

Methods

We used the publication patterns and dollar expenditures of VA-funded researchers to characterize the VA research portfolio by disease. We used health care utilization data from the VA for the same diseases to define veterans’ health needs. We then measured the level of correlation between the two and identified disease groups that were under- or over-represented in the research portfolio relative to disease expenditures. Finally, we used historic health care utilization trends combined with demographic projections to identify diseases and conditions that are increasing in costs and/or patient volume and consequently represent potential targets for future research investments.

Results

We found a significant correlation between research volume/expenditures and health utilization. Some disease groups were slightly under- or over-represented, but these deviations were relatively small. Diseases and conditions with the increasing utilization trend at the VA included hypertension, hypercholesterolemia, diabetes, hearing loss, sleeping disorders, complications of pregnancy, and several mental disorders.

Conclusions

Research investments at the VA are well aligned with veteran health needs. The VA can continue to meet these needs by supporting research on the diseases and conditions with a growing number of patients, costs of care, or both. Our approach can be used by other funders of disease research to characterize their portfolios and to plan research investments.     

Versatility in the acquisition of energy and carbon sources by the Apicomplexa

Members of the phylum Apicomplexa are motile and rapidly dividing intracellular parasites, able to occupy a large spectrum of niches by infecting diverse hosts and invading various cell types. As obligate intracellular parasites, most apicomplexans only survive for a short period extracellularly, and, during this time, have a high energy demand to power gliding motility and invasion into new host cells. Similarly, these fast‐replicating intracellular parasites are critically dependent on host‐cell nutrients as energy and carbon sources, noticeably for the extensive membrane biogenesis imposed during growth and division. To access host‐cell metabolites, the apicomplexans Toxoplasma gondii and Plasmodium falciparum have evolved strategies that exquisitely reflect adaptation to their respective niches. In the present review, we summarize and compare some recent findings regarding the energetic metabolism and carbon sources used by these two genetically tractable apicomplexans during host‐cell invasion and intracellular growth and replication.     

Versatility and continuity in the songs of thrushes Turdus spp.

A relationship between continuity and versatility was proposed by Hartshorne as a general feature of song organization in birds and argued to be linked to the avoidance of habituation (the monotony threshold principle). Thrushes of the genus Turdus vary greatly in song repertoire size, from the Redwing with a single song phrase to the Song Thrush which normally possesses well over 100 (Table 1), so that this group is well suited to looking for such a relationship. Recordings of 14 Turdus species were used to do so. Species in which successive songs do differ were indeed found to sing with shorter intervals between songs, though no equivalent relationship was found at the level of individual sound elements. There was a suggestion that species might fall into two contrasting groups without intermediates, one with continuous and highly varied songs and the other with discrete and simple songs. It is suggested that the relationship between continuity and versatility is a real phemomenon but that it is unlikely to be explained in the way that Hartshorne proposed. More likely is the hypothesis that continuous and varied songs have evolved primarily as mate attractants through sexual selection, while the principal role of discrete and simple songs is in communication between males.