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.     

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.     

Structural heterogeneity leads to functional homogeneity in A. marina phycocyanin

The major light harvesting antenna in all cyanobacterial species is the phycobilisome (PBS). The smallest PBS identified to date is that of Acaryochloris marina (A. marina), composed of a single four-hexamer rod. We have determined the crystal structure of phycocyanin (AmPC), the major component of the A. marina PBS (AmPBS) to 2.1?Å. The basic unit of the AmPC is a heterodimer of two related subunits (α and β), and we show that the asymmetric unit contains a superposition of two α and two β isoforms, the products of the simultaneous expression of different genes. This is the first time to our knowledge that isolated proteins crystallized with such identifiable heterogeneity. We believe that the presence of the different isoforms allows the AmPBS to have a significant bathochromic shift in its fluorescence emission spectrum, allowing, in the total absence of allophycocyanin, a better overlap with absorption of the chlorophyll d-containing reaction centers. We show that this bathochromic shift exists in intact AmPBS as well as in its disassembled components, thus suggesting that AmPC can efficiently serve as the AmPBS terminal emitter.     

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.     

Characterization of an archaeal multidrug transporter with a unique amino acid composition

The Smr family of multidrug transporters consists of small membrane proteins that extrude various drugs in exchange with protons rendering cells resistant to these drugs. Smr proteins identified to date have been found only in Eubacteria. In this work we present the cloning and characterization of an Smr protein from the archaeon Halobacterium salinarum, the first Smr in the archaeal kingdom. The protein, named Hsmr, was identified through sequence similarity to the Smr family, and the DNA sequence was cloned into an Escherichia coli expression system. Hsmr is heterologously expressed in a functional form despite the difference in lipid composition of the membrane and the lower salt in the cell and its environment. Cells harboring the Hsmr plasmid transport ethidium bromide in an uncoupler-sensitive process and gain resistance to ethidium bromide and acriflavine. Hsmr binds tetraphenylphosphonium (TPP(+)) with a relatively low affinity (K(D) approximately 200 nm) at low salt concentration that increases (K(D) approximately 40 nm) upon the addition of 2 m of either NaCl or KCl. The Hsmr protein contains many of the signature sequence elements of the Smr family and also a high content of negative residues in the loops, characteristic of extreme halophiles. Strikingly, Hsmr is composed of over 40% valine and alanine residues. These residues are clustered at certain regions of the protein in domains that are not important for activity, as judged from lack of conservation and from previous studies with other Smr proteins. We suggest that this high content of alanine and valine residues is a reflection of a "natural" alanine and valine scanning necessitated by the high GC content of the gene. This phenomenon reveals significant sequence elements in small multidrug transporters.