Proteolytic processing of QSOX1A ensures efficient secretion of a potent disulfide catalyst

QSOX1 (quiescin sulfhydryl oxidase 1) efficiently catalyses the insertion of disulfide bonds into a wide range of proteins. The enzyme is mechanistically well characterized, but its subcellular location and the identity of its protein substrates remain ill-defined. The function of QSOX1 is likely to involve disulfide formation in proteins entering the secretory pathway or outside the cell. In the present study, we show that this enzyme is efficiently secreted from mammalian cells despite the presence of a transmembrane domain. We identify internal cleavage sites and demonstrate that the protein is processed within the Golgi apparatus to yield soluble enzyme. As a consequence of this efficient processing, QSOX1 is probably functional outside the cell. Also, QSOX1 forms a dimer upon cleavage of the C-terminal domain. The processing of QSOX1 suggests a novel level of regulation of secretion of this potent disulfide catalyst and producer of hydrogen peroxide

Characterization of a cryogenic beam source for atoms and molecules

We present a combined experimental and theoretical study of beam formation from a cryogenic buffer gas cell. Atoms and molecules are loaded into the cell by laser ablation of a target, and are cooled and swept out of the cell by a flow of cold helium. We study the thermalization and flow dynamics inside the cell and measure how the speed, temperature, divergence and extraction efficiency of the beam are influenced by the helium flow. We use a finite element model to simulate the flow dynamics and use the predictions of this model to interpret our experimental results.Comment: 10 pages, 14 figure

A buffer gas beam source for short, intense and slow molecular pulses

Experiments with cold molecules usually begin with a molecular source. We describe the construction and characteristics of a cryogenic buff er gas source of CaF molecules. The source emits pulses with a typical duration of 240 μs, a mean speed of about 150 m/s, and a flux of 5x 10¹⁰ molecules per steradian per pulse in a single rotational state

Traveling wave deceleration of heavy polar molecules in low-field seeking states

We demonstrate the deceleration of heavy polar molecules in low-field seeking states by combining a cryogenic source and a travelling-wave Stark decelerator. The cryogenic source provides a high intensity beam with low speed and temperature, and the travelling-wave decelerator provides large deceleration forces and high phase-space acceptance. We prove these techniques using YbF molecules and find the experimental data to be in excellent agreement with numerical simulations. These methods extend the scope of Stark deceleration to a very wide range of molecules.Comment: 5 pages, 4 figure

Franck-Condon Factors and Radiative Lifetime of the A^{2}\Pi_{1/2} - X^{2}\Sigma^{+} Transition of Ytterbium Monoflouride, YbF

The fluorescence spectrum resulting from laser excitation of the A^{2}\Pi_{1/2} - X^{2}\Sigma^{+} (0,0) band of ytterbium monofluoride, YbF, has been recorded and analyzed to determine the Franck-Condon factors. The measured values are compared with those predicted from Rydberg-Klein-Rees (RKR) potential energy curves. From the fluorescence decay curve the radiative lifetime of the A^{2}\Pi_{1/2} state is measured to be 28\pm2 ns, and the corresponding transition dipole moment is 4.39\pm0.16 D. The implications for laser cooling YbF are discussed.Comment: 5 pages, 5 figure

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum

Background: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.Wellcome Trust [Wellcome 084812/Z/08/Z]; European Commission (EU FP7 Beta-Bat) [277713]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI-BIQ/119677/2010]info:eu-repo/semantics/publishedVersio