The Acidic Tail of the Cdc34 Ubiquitin-conjugating Enzyme Functions in Both Binding to and Catalysis with Ubiquitin Ligase SCFC^(dc4*)

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF^(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF^(Cdc4) and makes a major contribution to the submicromolar K_m of Cdc34 for SCF^(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly

Toward an Integrated Model of Capsule Regulation in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation

Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation

Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus—its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes

Cryptococcus neoformans Dual GDP-mannose transporters and their role in biology and virulence

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis

Development of a Simple Mechanical Screening Method for Predicting the Feedability of a Pharmaceutical FDM 3D Printing Filament

Purpose: The filament-based feeding mechanism employed by the majority of fused deposition modelling (FDM) 3D printers dictates that the materials must have very specific mechanical characteristics. Without a suitable mechanical profile, the filament can cause blockages in the printer. The purpose of this study was to develop a method to screen the mechanical properties of pharmaceutically-relevant, hot-melt extruded filaments to predetermine their suitability for FDM. Methods: A texture analyzer was used to simulate the forces a filament is subjected to inside the printer. The texture analyzer produced a force-distance curve referred to as the flexibility profile. Principal Component Analysis and Correlation Analysis statistical methods were then used to compare the flexibility profiles of commercial filaments to in-house made filaments. Results: Principal component analysis showed clearly separated clustering of filaments that suffer from mechanical defects versus filaments which are suitable for printing. Correlation scores likewise showed significantly greater values with feedable filaments than their mechanically deficient counterparts. Conclusion: The screening method developed in this study showed, with statistical significance and reproducibility, the ability to predetermine the feedability of extruded filaments into an FDM printer

Broad utility of an affinity-enrichment strategy for unanchored polyubiquitin chains

Protein ubiquitination is a common post-translational modification where selected targets are covalently modified by the ubiquitin protein, often in the form of isopeptide-linked polyubiquitin chains. More recently, unanchored (i.e. non-substrate-linked) polyubiquitin chains have also been described and implicated in a range of biological processes. The development of Tandem-repeated Ubiquitin-Binding Entities (TUBEs), engineered repeats of ubiquitin-binding domains that interact non-covalently with polyubiquitin, has allowed strategies for the affinity-enrichment of ubiquitinmodified proteins to be established, in some cases with linkage specificity. Here, we demonstrate the utility of a Free Ubiquitin-Binding Entity (FUBE), based on an ubiquitin-binding domain with high specificity for the free C-terminus of ubiquitin (the Znf-UBP domain of human USP5). In contrast to TUBEs which do not distinguish conjugated or free polyubiquitin, the FUBE exclusively recognises ubiquitin in its unconjugated form, including endogenous unanchored polyubiquitin chains. Affinity-enrichments using the FUBE demonstrate that unanchored polyubiquitin chains are present in different mammalian cell lines and accumulate when the 26S proteasome is pharmacologically inhibited, being retained on the proteasome. The high conservation of the ubiquitin sequence permits the FUBE to also be applied to the purification of endogenous unanchored polyubiquitin chains from species as diverse as Arabidopsis thaliana and Saccharomyces cerevisiae. The development and refinement of an affinity-enrichment strategy for unanchored polyubiquitin chains opens the way for more complete investigations into their biological significance. © 2013 Strachan J, et al

3D printing of five-in-one dose combination polypill with defined immediate and sustained release profiles

We have used three dimensional (3D) extrusion printing to manufacture a multi-active solid dosage form or so called polypill. This contains five compartmentalised drugs with two independently controlled and well-defined release profiles. This polypill demonstrates that complex medication regimes can be combined in a single personalised tablet. This could potentially improve adherence for those patients currently taking many separate tablets and also allow ready tailoring of a particular drug combination/drug release for the needs of an individual. The polypill here represents a cardiovascular treatment regime with the incorporation of an immediate release compartment with aspirin and hydrochlorothiazide and three sustained release compartments containing pravastatin, atenolol, and ramipril. X-ray powder diffraction (XRPD) and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) were used to assess drug-excipient interaction. The printed polypills were evaluated for drug release using USP dissolution testing. We found that the polypill showed the intended immediate and sustained release profiles based upon the active/excipient ratio used

Allelic Variation and Differential Expression of the mSIN3A Histone Deacetylase Complex Gene Arid4b Promote Mammary Tumor Growth and Metastasis

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT–rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA–mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer

3D inkjet printing of tablets exploiting bespoke complex geometries for controlled and tuneable drug release

A hot melt 3D inkjet printing method with the potential to manufacture formulations in complex and adaptable geometries for the controlled loading and release of medicines is presented. This first use of a precisely controlled solvent free inkjet printing to produce drug loaded solid dosage forms is demonstrated using a naturally derived FDA approved material (beeswax) as the drug carrier and fenofibrate as the drug. Tablets with bespoke geometries (honeycomb architecture) were fabricated. The honeycomb architecture was modified by control of the honeycomb cell size, and hence surface area to enable control of drug release profiles without the need to alter the formulation. Analysis of the formed tablets showed the drug to be evenly distributed within the beeswax at the bulk scale with evidence of some localization at the micron scale. An analytical model utilizing a Fickian description of diffusion was developed to allow the prediction of drug release. A comparison of experimental and predicted drug release data revealed that in addition to surface area, other factors such as the cell diameter in the case of the honeycomb geometry and material wettability must be considered in practical dosage form design. This information when combined with the range of achievable geometries could allow the bespoke production of optimized personalised medicines for a variety of delivery vehicles in addition to tablets, such as medical devices for example

HIF-1 and SKN-1 Coordinate the Transcriptional Response to Hydrogen Sulfide in Caenorhabditis elegans

Hydrogen sulfide (H2S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H2S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H2S effects physiological functions, we have measured transcriptional responses to H2S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H2S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H2S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H2S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H2S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H2S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H2S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H2S that culminates in a global reorganization of protein homeostasis networks