Model peptide‐based system used for the investigation of metal ions binding to histidine‐containing polypeptides

The reaction of histidine‐containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI–MS) and circular dichroism (CD). The analysis of CD‐spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal‐bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497–508, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The surfactant lipid transporter ABCA3 is N-terminally cleaved inside LAMP3-positive vesicles

ABCA3 mutations cause fatal surfactant deficiency and interstitial lung disease. ABCA3 protein is a lipid transporter indispensible for surfactant biogenesis and storage in lamellar bodies (LB). The protein folds in endoplasmic reticulum and is glycosylated in Golgi en route to the membrane of mature LB and their precursor multivesicular bodies (MVB). In immunoblots, C-terminally labeled ABCA3 appears as two protein bands of 150 and 190 kDa. Using N- and C-terminal protein tags and hindering ABCA3 processing we show that the 150 kDa protein represents the mature ABCA3 whose N-terminus is cleaved by a cysteine protease inside MVB/LB.

Structured summary

MINT-7996633: Calnexin (uniprotkb:P27824) and ABCA3 (uniprotkb:Q99758) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7996380, MINT-7996593, MINT-7996607: LAMP3 (uniprotkb:Q9UQV4) and ABCA3 (uniprotkb:Q99758) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Evidence for leptin receptor isoforms heteromerization at the cell surface

Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin’s effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

Structured summary

MINT-7714817: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by anti tag co-immunoprecipitation (MI:0007)MINT-7714785: LEPRc (uniprotkb:P48357-2) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714951, MINT-7714744: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714859: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714885, MINT-7714672: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by bioluminescence resonance energy transfer (MI:0012)MINT-7714835: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714914, MINT-7714723, MINT-7714759: LeprB (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714703, MINT-7714936, MINT-7714772: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714872: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by anti tag co-immunoprecipitation (MI:0007)     

A unique mechanism for the processive movement of single-headed myosin-IX

It has been puzzled that in spite of its single-headed structure, myosin-IX shows the typical character of processive motor in multi-molecule in vitro motility assay, because this cannot be explained by hand-over-hand mechanism of the two-headed processive myosins. Here, we show direct evidence of the processive movement of myosin-IX using two different single molecule techniques. Using optical trap nanometry, we found that myosin-IX takes several large ( approximately 20nm) steps before detaching from an actin filament. Furthermore, we directly visualized the single myosin-IX molecules moving on actin filaments for several hundred nanometers without dissociating from actin filament. Since myosin-IX processively moves without anchoring the neck domain, the result suggests that the neck tilting is not involved for the processive movement of myosin-IX. We propose that the myosin-IX head moves processively along an actin filament like an inchworm via a unique long and positively charged insertion in the loop 2 region of the head.     

Covalent labeling of cell-surface proteins for in-vivo FRET studies

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.     

Bioaccumulation of mercury in yellow perch (Perca flavescens) and common loons (Gavia immer) in relation to lake chemistry in Atlantic Canada

Mercury biomagnifies in aquatic foodwebs in freshwater lakes, and common loons (Gavia immer) breeding in eastern Canada can be exposed to reproductively toxic concentrations of mercury in their fish prey. We assessed the bioaccumulation and biomagnification of mercury in juvenile and adult common loons, and their preferred prey: yellow perch (Perca flavescens) in Kejimkujik National Park (KNP), Nova Scotia by measuring mercury levels and stable isotope ratios in tissues. Total mercury levels and stable-carbon (δ¹³C) and nitrogen isotope ratios (δ¹⁵N) were determined in composite whole-fish samples from lakes in KNP and blood samples from juvenile and adult loons captured on lakes in KNP and southern New Brunswick. Geometric mean mercury concentrations were 0.15 and 0.38 μg/g (wet wt.) in small (9-cm fork length) and large (17-cm fork length) yellow perch, and were 0.43 and 2.7 μg/g (wet wt.) in blood of juvenile and adult common loons, respectively. Mercury concentrations in perch and loons were positively associated with body mass and δ¹⁵N values. Juvenile loons and large yellow perch had similar mercury levels and δ¹⁵N values, indicating similar trophic status despite their 22-fold difference in body mass. Mercury concentrations were higher in yellow perch and common loons in acidic lakes. Our findings highlight the importance of both chemical and ecological factors in understanding mercury biomagnification in lakes and associated risks to fish-eating wildlife. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Use of hair tubes to survey for shrews: new methods for identification and quantification of abundance

• 1 Accurate and sensitive survey and monitoring methods are needed for shrews. We present a new design of hair tube and a new, simple method of species identification from multivariate analysis of four parameters measured from shrew guard hairs using a binocular microscope with incident light.
• 2 Multivariate analysis of these parameters measured from hairs of known identity showed that they can be used to identify hair to the species level with 85% accuracy.
• 3 We compared our indices of abundance from hair tubes (the hair tube index) with those from live trapping in 40 field margins. Capture‐mark‐recapture methods showed that capture rate did not vary systematically across sites, so that number of individuals captured was used as an index of abundance.
• 4 The hair tube index showed a significant association with the number of individuals captured for Sorex araneus and Neomys fodiens. The lack of a significant association for Sorex minutus may be because hair tubes are more sensitive in detecting this species than live trapping.
• 5 Hair tubes have additional advantages over live trapping, since they do not require frequent checking, are much lighter and cheaper than live traps, and no licence is required for their use in the UK. We therefore recommend consideration of their use in future surveys and monitoring studies of shrews. We provide an equation so that other researchers can use our multivariate method.

     

Potential of a synthetic aggregation pheromone for integrated pest management of Colorado potato beetle

Abstract

• 1 The relative number of colonizing adult Colorado potato beetles (CPB) Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) coming to pitfall traps baited with the aggregation pheromone (S)‐3,7‐dimethyl‐2‐oxo‐oct‐6‐ene‐1,3‐diol [(S)‐CPB I] and the use of the pheromone in a trap crop pest management strategy were evaluated in the field for the first time.
• 2 More than five‐fold more adult L. decemlineata were caught in pitfall traps baited with the pheromone compared with controls. However, attraction to the pheromone diminished after 5 days in the field.
• 3 In the trap crop management strategy, more colonizing adults were present in pheromone‐treated rows of potatoes compared with untreated middle rows.
• 4 Significantly fewer L. decemlineata egg masses and larvae were found in potato plots that were bordered by pheromone‐treated rows, or bordered by imidacloprid + pheromone‐treated rows, or rows treated at‐planting with imidacloprid compared with untreated (control) potato plots.
• 5 Densities of L. decemlineata egg masses and larvae and percentage defoliation were significantly lower, and marketable tuber yield significantly higher, in conventional imidacloprid‐treated potatoes compared with all other treatments.
• 6 Although our results demonstrate the potential for use of the aggregation pheromone in the management of L. decemlineata in the field, more research is needed to optimize the release rates of the attractant and incorporate control methods for cohabiting pests.

     

Comparison of the Paul ion trap to the linear ion trap for use in global proteomics

A critical evaluation of the performance of a 2-D linear ion trap (IT) instrument to two 3-D quadrupole IT instruments with emphasis on identification of rat serum proteins by bottom-up LC-MS/MS is presented. The speed and sensitivity of each of the instruments were investigated, and the effects that each of these have on the bottom-up proteomics identification approach are discussed.