Timed degradation of Mcl-1 controls mitotic cell death

Mitotic arrest can result in cell death through the process of apoptosis. We have shown by live-cell imaging that the ubiquitin-proteasome dependent proteolysis of the apoptotic regulator Mcl-1 under the control of the anaphase-promoting complex or cyclosome (APC/C) provides a timing mechanism that distinguishes prolonged mitotic arrest from normal mitosis

Mycobacterium tuberculosis type VII secretion system effectors differentially impact the ESCRT endomembrane damage response

Mycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity

Atypical APC/C-dependent degradation of Mcl-1 provides an apoptotic timer during mitotic arrest

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl-1 during an extended mitosis requires the anaphase-promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCF Using live-cell imaging, we show that the loss of Mcl-1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine-arginine (IR) C-terminal tail regulates the manner in which Mcl-1 engages with the APC/C, converting Mcl-1 from a Cdc20-dependent and checkpoint-controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl-1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl-1 degradation, providing an improved strategy to kill cancer cells

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

The Effect of Perilla frutescens extract on the oxidative stability of model food emulsions

The polyphenolic profile of leaves and stalks of Perilla frutescens, was assessed as a source of natural antioxidants. The amount of caffeic and rosmarinic acids, determined by high-performance liquid chromatography (HPLC), were 0.51 mg/g dry weight (DW) and 2.29 mg/g DW, respectively. The measurement of scavenging capacity against the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation, the oxygen radical absorbance capacity (ORAC), and the ferric reducing antioxidant power (FRAP) were 65.03 mg Trolox equivalents (TE)/g DW, 179.60 mg TE/g DW and 44.46 mg TE/g DW, respectively. P. frutescens extracts also showed good antioxidant properties in 10% sunflower oil-in-water emulsions during storage at 32 °C. Perilla extract at 320 ppm was as effective as butylated hydroxyanisole (BHA) at 20 ppm in slowing down the formation of hydroperoxides as measured by peroxide value, thiobarbituric acid reactive substances and hexanal content. The results of this study indicate that extract of P. frutescens may be suitable for use in the food matrix to help achieve potential health benefits

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

Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism

Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies

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