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The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor. [ABSTRACT FROM AUTHOR]

Cell‐surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram‐positive bacteria often contain autocatalytically formed covalent intramolecular cross‐links. While investigating the prevalence of such cross‐links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester‐bond‐containing proteins (TIE proteins). It has an N‐terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)‐like domains containing ester‐ or isopeptide‐bond cross‐links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs) and the Staphylococcal Superantigen-Like (SSL) exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX) have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac)-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect S. aureus from destruction in a whole blood killing (WBK) assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the S. aureus exotoxin expression (Sae) two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX) reveals that SElX is a unique single-domain SAg. In summary, SElX is an ‘SSL-like’ SAg. [ABSTRACT FROM AUTHOR]

The induction of Programmed Cell Death (PCD) requires the activation of complex responses involving the interplay of a variety of different cellular proteins, pathways, and processes. Uncovering the mechanisms regulating PCD requires an understanding of the different processes that both positively and negatively regulate cell death. Here we have examined the response of normal as well as PCD resistant yeast cells to different PCD inducing stresses. As expected cells expressing the pro-survival human 14-3-3β/α sequence show increased resistance to numerous stresses including copper and rapamycin. In contrast, other stresses including iron were more lethal in PCD resistant 14-3-3β/α expressing cells. The increased sensitivity to PCD was not iron and 14-3-3β/α specific since it was also observed with other stresses (hydroxyurea and zinc) and other pro-survival sequences (human TC-1 and H-ferritin). Although microscopical examination revealed little differences in morphology with iron or copper stresses, cells undergoing PCD in response to high levels of prolonged copper treatment were reduced in size. This supports the interaction some forms of PCD have with the mechanisms regulating cell growth. Analysis of iron-mediated effects in yeast mutant strains lacking key regulators suggests that a functional vacuole is required to mediate the synergistic effects of iron and 14-3-3β/α on yeast PCD. Finally, mild sub-lethal levels of copper were found to attenuate the observed inhibitory effects of iron. Taken together, we propose a model in which a subset of stresses like iron induces a complex process that requires the cross-talk of two different PCD inducing pathways. [ABSTRACT FROM AUTHOR]

Acute rheumatic fever (ARF) is an autoimmune response to Group A Streptococcus (GAS) infection. Repeated GAS exposures are proposed to 'prime' the immune system for autoimmunity. This notion of immune-priming by multiple GAS infections was first postulated in the 1960s, but direct experimental evidence to support the hypothesis has been lacking. Here, we present novel methodology, based on antibody responses to GAS T-antigens, that enables previous GAS exposures to be mapped in patient sera. T-antigens are surface expressed, type specific antigens and GAS strains fall into 18 major clades or T-types. A panel of recombinant T-antigens was generated and immunoassays were performed in parallel with serum depletion experiments allowing type-specific T-antigen antibodies to be distinguished from cross-reactive antibodies. At least two distinct GAS exposures were detected in each of the ARF sera tested. Furthermore, no two sera had the same T-antigen reactivity profile suggesting that each patient was exposed to a unique series of GAS T-types prior to developing ARF. The methods have provided much-needed experimental evidence to substantiate the immune-priming hypothesis, and will facilitate further serological profiling studies that explore the multifaceted interactions between GAS and the host. [ABSTRACT FROM AUTHOR]

The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength. [ABSTRACT FROM AUTHOR]

The chemical synthesis of analogue of a novel γ-secretase activating protein, which may play a pivotal role in the formation of amyloid peptides, the precursor to Alzheimer's disease, is described. The linear polypeptide sequence, consisting of 121 amino acids was assembled from four unprotected peptide building blocks using a convergent ligation-based synthesis. A strategic mutation of three glutamine residues to cysteine enabled the ligations, and the cysteines were subsequently converted to pseudoglutamines, to mimic the native glutamine. The full length unfolded protein was obtained in milligram amounts and was demonstrated to be homogeneous by liquid chromatography and mass spectrometry. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P21 and diffracted to 2.05 Å resolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases. [ABSTRACT FROM AUTHOR]

The pili expressed on the surface of the human pathogen Streptococcus pyogenes play an important role in host cell attachment, colonisation and pathogenesis. These pili are built from two or three components, an adhesin subunit at the tip, a major pilin that forms a polymeric shaft, and a basal pilin that is attached to the cell wall. Assembly is carried out by specific sortase (cysteine transpeptidase) enzyme. These components are encoded in a small gene cluster within the S. pyogenes genome, often together with another protein, SipA, whose function is unknown. We show through functional assays, carried out by expressing the S. pyogenes pilus components in Lactococcus lactis, SipA from the clinically important M1T1 strain is essential for pilus assembly, and that SipA function is likely to be conserved in all S. pyogenes. From the crystal structure of SipA we confirm that SipA belongs to the family of bacterial signal peptidases (SPases), which process the signal-peptides of secreted proteins. In contrast to a previous arm-swapped SipA dimer, this present structure shows that its principal domain closely resembles the catalytic domain of SPases and has a very similar peptide-binding cleft, but it lacks the catalytic Ser and Lys residues characteristic of SPases. In SipA these are replaced by Asp and Gly residues, which play no part in activity. We propose that SipA functions by binding a key component at the bacterial cell surface, in a conformation that facilitates pilus assembly. [ABSTRACT FROM AUTHOR]

Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, attachment to host cells, and pathogenesis. These proteins, and protein assemblies, such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such cross-links can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins. [ABSTRACT FROM AUTHOR]

RNA-dependent RNA polymerase activity is required for RNA interference ( RNAi) in many lower eukaryotes including the fission yeast Schizosacchromyces pombe. Together with Ago1 and Dcr1, the RNA-dependent RNA polymerase Rdp1 is critical for RNA-dependent transcriptional- and post-transcriptional gene silencing. Although the bulk of Rdp1 is localized to the nucleus, Ago1 and Dcr1 are primarily cytoplasmic. This may reflect the fact that Rdp1 is required early in the RNAi pathway to generate double strand RNA from transcripts that originate from centromeric loci. The relatively large size of Rdp1 (139.4 kD) precludes passive diffusion of the enzyme into the nucleus suggesting that karyopherin-dependent transport is involved in nuclear targeting of this enzyme. In this study, we report that the karyopherin/importin β3 homolog Sal3 is required for nuclear import of Rdp1 in S. pombe. Loss of nuclear Rdp1 was associated with substantially reduced transcriptional gene silencing, and surprisingly, post-transcriptional gene silencing which occurs in the cytoplasm of other eukaryotes, was also significantly affected. Together, these results identify Sal3 as a modulator of RNAi-dependent transcriptional gene silencing as well as a potential link between nuclear import and post-transcriptional gene silencing. [ABSTRACT FROM AUTHOR]

Following replication arrest the Cdc25 phosphatase is phosphorylated and inhibited by Cds1. It has previously been reported that expressing Cdc25 where 9 putative amino-terminal Cds1 phosphorylation sites have been substituted to alanine results in bypass of the DNA replication checkpoint. However, these results were acquired by expression of the phosphorylation mutant using a multicopy expression vector in a genetic background where the DNA replication checkpoint is intact. In order to clarify these results we constructed a Cdc25(9A)-GFP native promoter integrant and examined its effect on the replication checkpoint at endogenous expression levels. In this strain the replication checkpoint operates normally, conditional on the presence of the Mik1 kinase. In response to replication arrest the Cdc25(9A)-GFP protein is degraded, suggesting the presence of a backup mechanism to eliminate the phosphatase when it cannot be inhibited through phosphorylation. [ABSTRACT FROM AUTHOR]

Background: Expression of the HIV-1 vpr gene in human and fission yeast cells displays multiple highly conserved activities, which include induction of cell cycle G2 arrest and cell death. We have previously characterized a yeast heat shock protein 16 (Hsp16) that suppresses the Vpr activities when it is overproduced in fission yeast. Similar suppressive effects were observed when the fission yeast hsp16 gene was overexpressed in human cells or in the context of viral infection. In this study, we further characterized molecular actions underlying the suppressive effect of Hsp16 on the Vpr activities. Results: We show that the suppressive effect of Hsp16 on Vpr-dependent viral replication in proliferating T-lymphocytes is mediated through its C-terminal end. In addition, we show that Hsp16 inhibits viral infection in macrophages in a dose-dependent manner. Mechanistically, Hsp16 suppresses Vpr activities in a way that resembles the cellular heat shock response. In particular, Hsp16 activation is mediated by a heat shock factor (Hsf)-dependent mechanism. Interestingly, vpr gene expression elicits a moderate increase of endogenous Hsp16 but prevents its elevation when cells are grown under heat shock conditions that normally stimulate Hsp16 production. Similar responsive to Vpr elevation of Hsp and counteraction of this elevation by Vpr were also observed in our parallel mammalian studies. Since Hsf-mediated elevation of small Hsps occurs in all eukaryotes, this finding suggests that the anti-Vpr activity of Hsps is a conserved feature of these proteins. Conclusion: These data suggest that fission yeast could be used as a model to further delineate the potential dynamic and antagonistic interactions between HIV-1 Vpr and cellular heat shock responses involving Hsps. [ABSTRACT FROM AUTHOR]

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The article reports on the study that uses bacterial surface proteins attached to host cells through a mechanism used by immune system proteins that help eliminate invading pathogens in biological arms race.

This article presents the effective dielectric constant (εett) and attenuation construct (α) of an unshielded slot line and a coplanar strip line on a high-resistivity (5000-10,000 Ω-cm) silicon wafer. The εett (de to 40 GHz) and a (de to 26.5 GHz) in each case are determined from the measured resonant frequencies and insertion loss of a ring resonator. The measurements are carried out at room temperature. No dc bias is applied to the transmission lines. The attenuation for slot line and coplanar stripline on silicon are compared with microstrip line and coplanar waveguide on other semiconductor substrate materials. Lastly, a microstrip-to-slot-line transition and as use in a feed network for linearly tapered slot antennas is demonstrated. [ABSTRACT FROM AUTHOR]

Studies the effect of transforming growth factor-beta (TGF-beta) on bovine airway smooth muscle cells (BASMC) in vitro. Inhibitory and stimulatory effects of TGF-beta1; Increase in cell size due to TGF-beta1; Concentration of latent TGF-beta in the conditioned medium from the BASMC.

The minor pilin FctB is an integral part of the pilus assembly expressed by Streptococcus pyogenes. Since it is located at the cell wall, it can be hypothesized that it functions as a cell-wall anchor for the streptococcal pilus. In order to elucidate its structure, the genes for FctB from the S. pyogenes strains 90/306S and SF370 were cloned for overexpression in Escherichia coli. FctB from strain 90/306S was crystallized by the sitting-drop vapour-diffusion method using sodium citrate as a precipitant. The hexagonal FctB crystals belonged to space group P61 or P65, with unit-cell parameters a = b = 95.15, c = 100.25 Å, and diffracted to 2.9 Å resolution. [ABSTRACT FROM AUTHOR]

The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide bonds, provided the redox environment is controlled. The discovery of stabilizing isopeptide bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes. Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed isopeptide bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar β-clasp Ig-type domains. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the author discusses studies and best practices in after-school programming.