A 3-D in vitro co-culture model of mammary gland involution.

Involution is a process whereby the mammary gland undergoes extensive tissue remodelling involving exquisitely coordinated cell death, extracellular matrix degradation and adipose tissue regeneration following the weaning of offspring. These processes are mediated in part through Jak/Stat signalling pathways, which can be deregulated in breast cancer. Synthetic in vitro analogues of the breast could become important tools for studying tumorigenic processes, or as personalized drug discovery platforms and predictors of therapeutic response. Ideally, such models should support 3D neo-tissue formation, so as to recapitulate physiological organ function, and be compatible with high-throughput screening methodologies. We have combined cell lines of epithelial, stromal and immunological origin within engineered porous collagen/hyaluronic acid matrices, demonstrating 3D-specific molecular signatures. Furthermore seeded cells form mammary-like branched tissues, with lobuloalveolar structures that undergo inducible involution phenotypes reminiscent of the native gland under hormonal/cytokine regulation. We confirm that autophagy is mediated within differentiated mammary epithelial cells in a Stat-dependent manner at early time points following the removal of a prolactin stimulus (H/WD). In addition, epithelial cells express markers of an M2 macrophage lineage under H/WD, a process that is attenuated with the introduction of the monocyte/macrophage cell line RAW 264.7. Thus, such 3D models are suitable platforms for studying cell-cell interactions and cell death mechanisms in relation to cancer.This is the accepted manuscript. The final version is available from RSC at http://pubs.rsc.org/en/content/articlehtml/2014/ib/c3ib40257f

Structure and dynamics of a constitutively active neurotensin receptor

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist

Structural insight into mitochondrial β-barrel outer membrane protein biogenesis.

In mitochondria, -barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold -barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane -barrel with a single polypeptide-transport associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 barrel opens a lateral gate to accommodate its substrates

Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish

The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with superassembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here, we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2 + IV2, I + IV, and I + III2 + IV2. To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (scaf1). scaf1 / fish displayed altered OXPHOS activity due to the disrupted interaction of complexes III and IV. scaf1 / fish were smaller in size and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by Scaf1 modulates OXPHOS efficiency and allows the optimization of metabolic resources.Microscopy Imaging Center of the University of BernSpanish Ministry of Economy and Competitiveness, MINECO SAF2015-65633-RSpanish Ministry of Economy and Competitiveness, MINECO SAF2015-65633-RHuman Frontier Science Program RGP0016/2018European Research Council (ERC) 337703SNF 31003A-159721Swiss National Science Foundation (SNSF) 320030_170062MINECO BIO2015-67580-PCarlos III Institute of Health-Fondo de Investigacion Sanitaria) PRB3 IPT17/0019Fundacion La Marato TV3La Caixa Foundation HR17-00247Ministry of Economy, Industry and Competitiveness (MEIC)Pro-CNIC FoundationSevero Ochoa Center of Excellence (MEIC award) SEV-2015-050

Grifonin-1: A Small HIV-1 Entry Inhibitor Derived from the Algal Lectin, Griffithsin

Background: Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties. Methodology/Results: The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC50 of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface. Conclusion: Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1

Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase

TLR3, TLR7, and TLR9 are cleaved in the same step-wise manner in all immune cell types examined

Structural insight into mitochondrial β-barrel outer membrane protein biogenesis

Abstract: In mitochondria, β-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane β-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 β-barrel opens a lateral gate to accommodate its substrates

An essential role for the N-terminal fragment of Toll-like receptor 9 in DNA sensing

Toll-like receptor 9 (TLR9) is an innate immune sensor for microbial DNA that erroneously responds to self DNA in autoimmune disease. To prevent autoimmune responses, Toll-like receptor 9 is excluded from the cell surface and silenced until the N-terminal half of the ectodomain (TLR9N) is cleaved off in the endolysosome. Truncated Toll-like receptor 9 (TLR9C) senses ingested microbial DNA, although the precise role of the truncation remains controversial. Here we show that TLR9 is expressed on the surface of splenic dendritic cells. Following the cleavage of TLR9 in the endolysosome, N-terminal half of the ectodomain remains associated with truncated TLR9, forming the complex TLR9N + C. The TLR9-dependent cytokine production by Tlr9(-/-) dendritic cells is rescued by a combination of TLR9N and TLR9C, but not by TLR9C alone. These results demonstrate that the TLR9N + C complex is a bona fide DNA sensor

Sap Transporter Mediated Import and Subsequent Degradation of Antimicrobial Peptides in Haemophilus

Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides