Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.     

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.     

IL-17 and FGF signaling involved in mouse mesenchymal stem cell proliferation

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin 17 (IL-17) and basic fibroblast growth factor (bFGF), on BM-MSCs growth and elucidated the signaling pathways involved. The results revealed that IL-17 increased the frequency of colony-forming units fibroblast (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-17 and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-17 and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.     

Structure and Mechanism of Iron Translocation by a Dps Protein from Microbacterium arborescens

Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.     

Noncanonical DNA elements in the lamin B2 origin of DNA replication

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mm Tris base, 89 mm boric acid, and 2 mm MgCl(2)) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT)(40) do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads.     

Mechanism of polymerase collision release from sliding clamps on the lagging strand

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the ‘collision release’ model, the lagging strand polymerase collides with the 5′ terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5′ terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA‐binding elements are involved, the τ subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The τ subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.     

Effects of Long-Term Pioglitazone Treatment on Peripheral and Central Markers of Aging

Background

Thiazolidinediones (TZDs) activate peroxisome proliferator-activated receptor gamma (PPARγ) and are used clinically to help restore peripheral insulin sensitivity in Type 2 diabetes (T2DM). Interestingly, long-term treatment of mouse models of Alzheimer''s disease (AD) with TZDs also has been shown to reduce several well-established brain biomarkers of AD including inflammation, oxidative stress and Aβ accumulation. While TZD''s actions in AD models help to elucidate the mechanisms underlying their potentially beneficial effects in AD patients, little is known about the functional consequences of TZDs in animal models of normal aging. Because aging is a common risk factor for both AD and T2DM, we investigated whether the TZD, pioglitazone could alter brain aging under non-pathological conditions.

Methods and Findings

We used the F344 rat model of aging, and monitored behavioral, electrophysiological, and molecular variables to assess the effects of pioglitazone (PIO-Actos® a TZD) on several peripheral (blood and liver) and central (hippocampal) biomarkers of aging. Starting at 3 months or 17 months of age, male rats were treated for 4–5 months with either a control or a PIO-containing diet (final dose approximately 2.3 mg/kg body weight/day). A significant reduction in the Ca²⁺-dependent afterhyperpolarization was seen in the aged animals, with no significant change in long-term potentiation maintenance or learning and memory performance. Blood insulin levels were unchanged with age, but significantly reduced by PIO. Finally, a combination of microarray analyses on hippocampal tissue and serum-based multiplex cytokine assays revealed that age-dependent inflammatory increases were not reversed by PIO.

Conclusions

While current research efforts continue to identify the underlying processes responsible for the progressive decline in cognitive function seen during normal aging, available medical treatments are still very limited. Because TZDs have been shown to have benefits in age-related conditions such as T2DM and AD, our study was aimed at elucidating PIO''s potentially beneficial actions in normal aging. Using a clinically-relevant dose and delivery method, long-term PIO treatment was able to blunt several indices of aging but apparently affected neither age-related cognitive decline nor peripheral/central age-related increases in inflammatory signaling.     

Early response to tendon fatigue damage accumulation in a novel in vivo model

This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1–35 N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.     

C5orf42 is the major gene responsible for OFD syndrome type VI

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the “Joubert syndrome related disorders”. Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.     

Organic Cultivation of Triticum turgidum subsp. durum Is Reflected in the Flour-Sourdough Fermentation-Bread Axis

Triticum turgidum subsp. durum was grown according to four farming systems: conventional (C_ONV), organic with cow manure (O_MAN) or green manure (O_LEG), and without inputs (NO_INPUT). Some chemical and technological characteristics differed between C_ONV and organic flours. As shown by two-dimensional electrophoresis (2-DE) analysis, O_MAN and O_LEG flours showed the highest number of gliadins, and O_MAN flour also had the highest number of high-molecular-mass glutenins. Type I sourdoughs were prepared at the laboratory level through a back-slopping procedure, and the bacterial ecology during sourdough preparation was described by 16S rRNA gene pyrosequencing. Before fermentation, the dough made with C_ONV flour showed the highest bacterial diversity. Flours were variously contaminated by genera belonging to the Proteobacteria, Firmicutes, and Actinobacteria. Mature sourdoughs were completely and stably dominated by lactic acid bacteria. The diversity of Firmicutes was the highest for mature sourdoughs made with organic and, especially, NO_INPUT flours. Beta diversity analysis based on the weighted UniFrac distance showed differences between doughs and sourdoughs. Those made with C_ONV flour were separated from the other with organic flours. Lactic acid bacterium microbiota structure was qualitatively confirmed through the culturing method. As shown by PCR-denaturing gradient gel electrophoresis (DGGE) analysis, yeasts belonging to the genera Saccharomyces, Candida, Kazachstania, and Rhodotorula occurred in all sourdoughs. Levels of bound phenolic acids and phytase and antioxidant activities differed depending on the farming system. Mature sourdoughs were used for bread making. Technological characteristics were superior in the breads made with organic sourdoughs. The farming system is another determinant affecting the sourdough microbiota. The organic cultivation of durum wheat was reflected along the flour-sourdough fermentation-bread axis.