Sec22b is a critical and nonredundant regulator of plasma cell maintenance.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells.

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis of breast cancer cells. Basement membrane degradation is mediated by targeted secretion of various matrix metalloproteinases (MMPs). Specifically, MMP2 and MMP9 (MMP2/9) possess the ability to hydrolyze components of the basement membrane and regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates targeting of MMP2/9 to the invadopodia during cancer cell invasion remains to be defined. Because Rab GTPases are key regulators of membrane transport, we screened a human Rab siRNA library and identified Rab40b GTPase as a protein required for secretion of MMP2/9. We also have shown that Rab40b functions during at least two distinct steps of MMP2/9 transport. Here, we demonstrate that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. We also show that Rab40b regulates transport of MMP2/9 secretory vesicles during invadopodia formation and is required for invadopodia-dependent extracellular matrix degradation. Finally, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. On the basis of these findings, we propose that Rab40b mediates trafficking of MMP2/9 during invadopodia formation and metastasis of breast cancer cells.

Identification of the wheat seed protein CM3 as a highly active emulsifier using a novel functional screen.

Lyophilized albumin protein fractions were prepared from flour of four varieties of wheat: Triticum aestivum cvs. Mercia and Riband, Triticum aestivum var. spelta, and Triticum turgidum var. durum (Kamut). The dry powders were redissolved in sodium phosphate buffers at pH 3.0, 6.5, or 8.0 and at ionic strengths of 0.1 or 1.0 M to a concentration of 0.1% (w/v). Emulsions formed by sonication of protein solutions with n-hexadecane were aged at room temperature and separated into aqueous, interstitial, and interfacial phases. The distinct emulsion components were lyophilized and analyzed by RP-HPLC. A protein was observed to be preferentially located in the interfacial component and subsequently purified from a total albumin fraction and identified by N-terminal sequencing as CM3, an alpha-amylase inhibitor subunit. Measurement of the equilibrium surface tension of CM3 as a function of protein concentration demonstrated that it was at least as active as bovine beta-lactoglobulin, an established protein emulsifier. Furthermore, measurement of the surface dilational elastic modulus at an air/water interface demonstrated the formation of a viscoelastic film, while fluorescence and FT-IR spectroscopic measurements on adsorbed and nonadsorbed CM3 suggest that the secondary structure is essentially unchanged upon adsorption to an oil/water interface. It is concluded that functional screening is a valid approach to identify novel protein emulsifiers in complex mixtures.

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking.

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.

Study of wheat high molecular weight 1Dx5 subunit by (13)C and (1)H solid-state NMR. II. Roles of nonrepetitive terminal domains and length of repetitive domain.

This work follows a previous article that addressed the role of disulfide bonds in the behavior of the 1Dx5 subunit upon hydration. Here the roles of nonrepetitive terminal domains present and the length of the central repetitive domain in the hydration of 1Dx5 are investigated. This was achieved by comparing the hydration behavior of suitable model samples determined by (13)C- and (1)H-NMR: an alkylated 1Dx5 subunit (alk1Dx5), a recombinant 58-kDa peptide corresponding to the central repetitive domain of 1Dx5 (i.e., lacking the terminal domains), and two synthetic peptides (with 6 and 21 amino acid residues) based on the consensus repeat motifs of the central domain. The (13)C cross-polarization and magic angle spinning (MAS) experiments recorded as a function of hydration gave information about the protein or peptide fractions resisting plasticization. Conversely, (13)C single pulse excitation and (1)H-MAS gave information on the more plasticized segments. The results are consistent with the previous proposal of a hydrated network held by hydrogen-bonded glutamines and possibly hydrophobic interactions. The nonrepetitive terminal domains were found to induce water insolubility and a generally higher network hindrance. Shorter chain lengths were shown to increase plasticization and water solubility. However, at low water contents, the 21-mer peptide was characterized by higher hindrance in the megahertz and kilohertz frequency ranges compared to the longer peptide; and a tendency for a few hydrogen-bonded glutamines and hydrophobic residues to remain relatively hindered was still observed, as for the protein and large peptide. It is suggested that this ability is strongly dependent on the peptide primary structure.