Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold

Architectures and biogenesis of non-flagellar protein appendages in Gram-negative bacteria

Bacteria commonly expose non-flagellar proteinaceous appendages on their outer surfaces. These extracellular structures, called pili or fimbriae, are employed in attachment and invasion, biofilm formation, cell motility or protein and DNA transport across membranes. Over the past 15 years, the power of molecular and structural techniques has revolutionalized our understanding of the biogenesis, structure, function and mode of action of these bacterial organelles. Here, we review the five known classes of Gram-negative non-flagellar appendages from a biosynthetic and structural point of view

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

BACKGROUND: Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS: We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS: We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION: We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature

[18F]Nifene test–retest reproducibility in first‐in‐human imaging of α4β2* nicotinic acetylcholine receptors

The aim of this study was to examine the suitability of [18 F]nifene, a novel α4β2* nicotinic acetylcholine receptor (nAChR) radiotracer, for in vivo brain imaging in a first-in-human study. Eight healthy subjects (4 M,4 F;21-69,44 ± 21 yrs) underwent a [18 F]nifene positron emission tomography scan (200 ± 3.7 MBq), and seven underwent a second scan within 58 ± 31 days. Regional estimates of DVR were measured using the multilinear reference tissue model (MRTM2) with the corpus callosum as reference region. DVR reproducibility was evaluated with test-retest variability (TRV) and intraclass correlation coefficient (ICC). The DVR ranged from 1.3 to 2.5 across brain regions with a TRV of 0-7%, and did not demonstrate a systematic difference between test and retest. The ICCs ranged from 0.2 to 0.9. DVR estimates were stable after 40 min. The binding profile and tracer kinetics of [18 F]nifene make it a promising α4β2* nAChR radiotracer for scientific research in humans, with reliable DVR test-retest reproducibility

Measuring α4β2∗ Nicotinic Acetylcholine Receptor Density in Vivo with [18F]nifene PET in the Nonhuman Primate

[(18)F]Nifene is an agonist PET radioligand developed to image α4β2* nicotinic acetylcholine receptors (nAChRs). This work aims to quantify the receptor density (Bmax) of α4β2* nAChRs and the in vivo (apparent) dissociation constant (KDapp) of [(18)F]nifene. Multiple-injection [(18)F]nifene experiments with varying cold nifene masses were conducted on four rhesus monkeys with a microPET P4 scanner. Compartment modeling techniques were used to estimate regional Bmax values and a global value of KDapp. The fast kinetic properties of [(18)F]nifene also permitted alternative estimates of Bmax and KDapp at transient equilibrium with the same experimental data using Scatchard-like methodologies. Averaged across subjects, the compartment modeling analysis yielded Bmax values of 4.8±1.4, 4.3±1.0, 1.2±0.4, and 1.2±0.3 pmol/mL in the regions of antereoventral thalamus, lateral geniculate, frontal cortex, and subiculum, respectively. The KDapp of nifene was 2.4±0.3 pmol/mL. The Scatchard analysis based on graphical evaluation of the data after transient equilibrium yielded Bmax estimations comparable to the modeling results with a positive bias of 28%. These findings show the utility of [(18)F]nifene for measuring α4β2* nAChR Bmax in vivo in the rhesus monkey with a single PET experiment