Quinolinic Acid Amyloid-like Fibrillar Assemblies Seed α-Synuclein Aggregation

Quinolinic acid (QA), a downstream neurometabolite in the kynurenine pathway, the biosynthetic pathway of tryptophan, is associated with neurodegenerative diseases pathology. Mutations in genes encoding kynurenine pathway enzymes, which control the level of QA production, are linked with elevated risk of developing Parkinson's disease. Recent findings have revealed the accumulation and deposition of QA in post-mortem samples, as well as in cellular models of Alzheimer's disease and related disorders. Furthermore, intrastriatal inoculation of mice with QA results in increased levels of phosphorylated α-synuclein and neurodegenerative pathological and behavioral characteristics. However, the cellular and molecular mechanisms underlying the involvement of QA accumulation in protein aggregation and neurodegeneration remain elusive. We recently established that self-assembled ordered structures are formed by various metabolites and hypothesized that these “metabolite amyloids” may seed amyloidogenic proteins. Here we demonstrate the formation of QA amyloid-like fibrillar assemblies and seeding of α-synuclein aggregation by these nanostructures both in vitro and in cell culture. Notably, α-synuclein aggregation kinetics was accelerated by an order of magnitude. Additional amyloid-like properties of QA assemblies were demonstrated using thioflavin T assay, powder X-ray diffraction and cell apoptosis analysis. Moreover, fluorescently labeled QA assemblies were internalized by neuronal cells and co-localized with α-synuclein aggregates. In addition, we observed cell-to-cell propagation of fluorescently labeled QA assemblies in a co-culture of treated and untreated cells. Our findings suggest that excess QA levels, due to mutations in the kynurenine pathway, for example, may lead to the formation of metabolite assemblies that seed α-synuclein aggregation, resulting in neuronal toxicity and induction of Parkinson's disease.     

Long-term balancing selection at the antiviral gene OAS1 in Central African chimpanzees

Oligoadenylate synthetases (OAS) are interferon-induced enzymes that participate in the first line of defense against a wide range of viral infection in animals. Upon activation by viral double-stranded RNA, OAS synthesizes (2-5) oligoadenylates, which activate RNase L, leading to the nonspecific degradation of cellular and viral RNA. Some association studies in humans suggest that variation at one of the OAS genes, OAS1, could be influencing host susceptibility to viral infection. We assessed the diversity of OAS1 in hominoid primates with a focus on chimpanzees. We found that the OAS1 gene is extremely polymorphic in Central African chimpanzee and exhibits levels of silent and replacement diversity much higher than neutral regions of the chimpanzee genome. This level of variation strongly suggests that balancing selection is acting on OAS1, and indeed, this conclusion was validated by several tests of neutrality. We further demonstrated that balancing selection has been acting at this locus since the split between chimpanzees, humans, and gorillas (~8.6 Ma) and caused the persistence of two deeply divergent allelic lineages in Central African chimpanzees. These two groups of OAS1 alleles differ by a large number of amino acids (a.a.), including several a.a. putatively involved in RNA binding. It is therefore very likely that variation at the OAS1 locus affects the innate immune response of individuals to specific viral infection. Our data strongly suggest that interactions between viral RNA and OAS1 are responsible for the maintenance of ancestral polymorphisms at this locus for at least 13.2 My.     

Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

We applied the patch-clamp technique to investigate the transport properties of the Slow Vacuolar (SV) channel identified in leaf vacuoles of Alyssum bertolonii Desv., a nickel hyperaccumulator plant growing in serpentine soil of the northern Apennines (Italy). SV currents recorded in vacuoles from adult plants collected in their natural habitat showed high sensitivity towards cytosolic nickel. Dose-response analyses indicated half-maximal current inhibition at submicromolar concentrations, i.e. up to three orders of magnitude lower than previously reported values from other plant species. The voltage-dependent increase of residual currents at saturating nickel concentrations could be interpreted as relief of channel block by nickel permeation at high positive membrane potentials. Including young plants of A. bertolonii into the study, we found that SV channels from these plants did not display elevated nickel sensitivity. This difference may be related to age-dependent changes in nickel hyperaccumulation of A. bertolonii leaf cells.     

The polyembryonic wasp Copidosoma floridanum produces two castes by differentially parceling the germ line to daughter embryos during embryo proliferation

Eggs of the polyembryonic wasp Copidosoma floridanum undergo a clonal phase of proliferation, which results in the formation of thousands of embryos called secondary morulae and two castes called reproductive and soldier larvae. C. floridanum establishes the germ line early in development, and prior studies indicate that embryos with primordial germ cells (PGCs) develop into reproductive larvae while embryos without PGCs develop into soldiers. However, it is unclear how embryos lacking PGCs form and whether all or only some morulae contribute to the proliferation process. Here, we report that most embryos lacking PGCs form by division of a secondary morula into one daughter embryo that inherits the germ line and another that does not. C. floridanum embryos also incorporate 5-bromo-2′-deoxyuridine (BrdU), which allows PGCs and other cell types to be labeled during the S phase of the cell cycle. Continuous BrdU labeling indicated that all secondary morulae cycle during the proliferation phase of embryogenesis. Double labeling with BrdU and the mitosis marker anti-phospho-histone H3 indicated that the median length of the G2 phase of the cell cycle was 18 h with a minimum duration of 4 h. Mitosis of PGCs and presumptive somatic stem cells in secondary morulae was asynchronous, but cells of the inner membrane exhibited synchronous mitosis. Overall, our results suggest that all secondary morulae contribute to the formation of new embryos during the proliferation phase of embryogenesis and that PGCs are involved in regulating both proliferation and caste formation.     

Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

Morphogenesis of the developing mammary gland: Stage-dependent impact of adipocytes

Mammary gland development is critically dependent on the interactions between the stromal and the epithelial compartments within the gland. These events are under the control of a complex interplay of circulating and locally acting hormones and growth factors. To analyze the temporal and quantitative contributions of stromal adipocytes, we took advantage of the FAT-ATTAC mice (apoptosis through triggered activation of caspase-8), a model of inducible and reversible loss of adipocytes. This loss can be achieved through the induced dimerization of a caspase-8 fusion protein. In the context of female mice, we can achieve ablation of mammary adipocytes relatively selectively without affecting other fat pads. Under these conditions, we find that adipocytes are essential for the formation of the extended network of ducts in the mammary gland during puberty. Beyond their role in development, adipocytes are also essential to maintain the normal alveolar structures that develop during adulthood. Loss of adipose tissue initiated 2 weeks after birth triggers fewer duct branching points and fewer terminal end buds (TEBs) and also triggers changes in proliferation and apoptosis in the epithelium associated with the TEBs. The reduced developmental pace that adipocyte-ablated glands undergo is reversible, as the emergence of new local adipocytes, upon cessation of treatment, enables the ductal epithelium to resume growth. Conversely, loss of local adipocytes initiated at 7 weeks of age resulted in excessive lobulation, indicating that adipocytes are critically involved in maintaining proper architecture and functionality of the mammary epithelium. Collectively, using a unique model of inducible and reversible loss of adipocytes, our observations suggest that adipocytes are required for proper development during puberty and for the maintenance of the ductal architecture in the adult mammary gland.     

Decidual NK cells regulate key developmental processes at the human fetal-maternal interface

Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.     

The membrane topology of EmrE - a small multidrug transporter from Escherichia coli

EmrE is a multidrug transporter from Escherichia coli that belongs to the Smr family of small multidrug transporters. The secondary structure of EmrE consists of a four helical bundle, as judged by different techniques. EmrE has been extensively characterized; nevertheless, the membrane topology of EmrE has not been determined yet. Previous work with a homologous Smr protein provided partial information of the membrane topology, however the location of the carboxy-terminus remained inconclusive. In this work we probed the membrane topology of EmrE, focusing on the carboxy-terminus of the protein, using two independent approaches. Our results support a secondary structure where the carboxy-terminus faces the cytoplasm, while the first loop faces the periplasm.