Human adrenoleukodystrophy protein and related peroxisomal ABC transporters interact with the peroxisomal assembly protein PEX19p

Four ABC half transporters (ALDP, ALDRP, PMP70, and PMP69) have been identified in the mammalian peroxisomal membrane but no function has been unambiguously assigned to any of them. To date X-linked adrenoleukodystrophy (X-ALD) is the only human disease known to result from a defect of one of these ABC transporters, ALDP. Using the yeast two-hybrid system and in vitro GST pull-down assays, we identified the peroxin PEX19p as a novel interactor of ALDP, ALDRP, and PMP70. The cytosolic farnesylated protein PEX19p was previously shown to be involved in an early step of the peroxisomal biogenesis. The PEX19p interaction occurs in an internal N-terminal region of ALDP which we verified to be important for proper peroxisomal targeting of this protein. Farnesylated wild-type PEX19p and a farnesylation-deficient mutant PEX19p did not differ in their ability to bind to ALDP. Our data provide evidence that PEX19p is a cytosolic acceptor protein for the peroxisomal ABC transporters ALDP, PMP70, and ALDRP and might be involved in the intracellular sorting and trafficking of these proteins to the peroxisomal membrane.     

The chemistry of rhenium and technetium. Part III. The synthesis and characterisation of oxo-bis-(tetrathiomolybdenato)-technetium(V) and its reduction by triphenylphosphine

The Complexes (Bu₄N)[TcO(MoS₄)₂] and Tc- (PPh₃)₂(MoS₄)₂ were prepared. The former complex has a much lower Tc-O stretching frequency than is generally found gor the TcO³⁺. moiety. The latter technetium(IV) Complex was obtained by the reduction of Tc^(v) O(MoS₄)₂^? with triphenylphosphine and also by the substitution reaction of TcCl₄(PPh₃)2 with MoS₄^2-. Previous reductions of this nature have led to the isolation of species that differ by two formal oxidation state numbers from the oxidant.     

Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large‐scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co‐evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.     

Direct Transfer of Viral and Cellular Proteins from Varicella-Zoster Virus-Infected Non-Neuronal Cells to Human Axons

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.     

Topical Delivery of 5-Fluorouracil from Pheroid™ Formulations and the In Vitro Efficacy Against Human Melanoma

Drug delivery vehicles can influence the topical delivery and the efficacy of an active pharmaceutical ingredient (API). In this study, the influence of Pheroid™ technology, which is a unique colloidal drug delivery system, on the skin permeation and antimelanoma efficacy of 5-fluorouracil were investigated. Lotions containing Pheroid™ with different concentrations of 5-fluorouracil were formulated then used in Franz cell skin diffusion studies and tape stripping. The in vitro efficacy of 5-fluorouracil against human melanoma cells (A375) was investigated using a flow cytometric apoptosis assay. Statistically significant concentrations of 5-fluorouracil diffused into and through the skin with Pheroid™ formulations resulting in an enhanced in vitro skin permeation from the 4.0% 5-fluorouracil lotion (p < 0.05). The stratum corneum-epidermis and epidermis-dermis retained 5-fluorouracil concentrations of 2.31 and 6.69 μg/ml, respectively, after a diffusion study with the 4.0% Pheroid™ lotion. Subsequent to the apoptosis assay, significant differences were observed between the effect of 13.33 μg/ml 5-fluorouracil in Pheroid™ lotion and the effects of the controls. The results obtained suggest that the Pheroid™ drug delivery system possibly enhances the flux and delivery of 5-fluorouracil into the skin. Therefore, using Pheroid™ could possibly be advantageous with respect to topical delivery of 5-fluorouracil.KEY WORDS: A375 cells, cell culture, flow cytometry, melanoma, permeation enhancer     

Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1

Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets.The coordination of cell growth and division represents a fundamental concept in cell biology. The mechanisms that promote polarized growth and drive cell cycle progression are complex signaling networks that operate in a wide range of cell types and organisms. Understanding these networks and their molecular connections requires large-scale approaches that define the underlying biochemical reactions. Phosphorylation drives many events in both cell polarity and cell cycle signaling, and protein kinases that act in both processes represent key players in coordinated growth and division.The fission yeast S. pombe has served as a long-standing model organism for studies on cell polarity and the cell cycle. The fission yeast protein kinase Pom1 is an intriguing candidate to function in the coordination of polarized growth and cell cycle progression. This DYRK¹ family kinase was originally identified as a polarity mutant (hence the name Pom1) in a genetic screen for misshapen cells (1). Later studies revealed an additional role for Pom1 in cell cycle progression, where it delays mitotic entry until cells reach a critical size threshold (2, 3). Thus, pom1Δ mutant cells display defects in both cell polarity and cell size at mitosis, as well as misplaced division septa (1–6). Mutations that impair Pom1 kinase activity mimic these deletion phenotypes, indicating a key role for Pom1-dependent phosphorylation. The pleiotropic phenotype of pom1 mutants might result from Pom1 phosphorylating distinct substrates for cell polarity versus mitotic entry, but the targets of Pom1 kinase activity are largely unknown. Only two Pom1 substrates have been identified to date. First, Pom1 auto-phosphorylates as part of a mechanism that promotes localization in a cortical gradient enriched at cell tips (7). Second, Pom1 phosphorylates two regions of the protein kinase Cdr2. Phosphorylation of Cdr2 C terminus is proposed to prevent mitotic entry by inhibiting Cdr2 kinase activity (8, 9), while phosphorylation near membrane-binding motifs of Cdr2 promotes medial cell division by inhibiting localization of Cdr2 at cell tips (10). It has been unclear if Cdr2 represents the only cell cycle target of Pom1 kinase activity, and no cell polarity targets of Pom1 have been identified. In order to clarify how this protein kinase controls multiple cellular processes, we have comprehensively cataloged Pom1 substrates by quantitative phosphoproteomics. Such a large-scale approach also has the potential to reveal general mechanisms that operate in the coordination of cell growth and division.Stable isotope labeling of amino acids in culture (SILAC) combined with phosphopeptide enrichment and mass spectrometry has allowed the proteome-wide analysis of protein phosphorylation from diverse experimental systems (11–15). In this approach, cells are grown separately in media containing normal (“light”) or isotope-labeled (“heavy”) arginine and lysine, treated, mixed, and processed for LC-MS/MS analysis. In combination with analog-sensitive protein kinase mutants, which can be rapidly and specifically inhibited by nonhydrolyzable ATP analogs (16, 17), SILAC presents a powerful approach to identify cellular phosphorylation events that depend on a specific protein kinase. This method is particularly well suited for studies in yeast, where analog-sensitive protein kinase mutants can be readily integrated into the genome.In this study, we have employed SILAC-based phosphoproteomics to identify Pom1 substrates in fission yeast. New Pom1 targets were verified as direct substrates in vitro, and our analysis indicates that Pom1 controls cell cycle progression through a single target while coordinating a more complex network of cell polarity targets.     

Integrated Microfluidics for Protein Modification Discovery

Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system''s potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.Protein post-translational modifications (PTMs)¹ vastly diversify eukaryotic proteomes and are integrated in essentially all cellular processes (1). Proteomic approaches, such as mass spectrometry (MS), have been instrumental in monitoring global molecular dynamics for research and clinical applications (2–5). However, even in this modern era, large-scale analyses of PTMs by MS is challenging because of the limited number of modified peptides derived from proteins that, by themselves, may not be abundant. Moreover, comprehensive PTM analysis by MS often requires significant amounts of biological material that may not be available. PTM analysis using protein arrays can overcome these limitations because of the equimolar amount of the arrayed proteins (6, 7). Large-scale protein arrays have been successfully integrated into PTM research (8, 9). However, this technology relies on pre-purified proteins that are arrayed on a surface and thus, incompatible with biochemically challenging proteins, let alone insoluble proteins. Moreover, the production of recombinant protein arrays is impractical in-house. Therefore, such arrays cannot be used fresh, and they are inherently limited to certain designs, protein compositions, and model organisms of high commercial value. To overcome the abovementioned limitations, we designed a modular integrated microfluidic platform for PTM analysis (IMPA).