Inadequate BiP availability defines endoplasmic reticulum stress.

How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible bulk expression of secretory immunoglobulin M heavy chain (μs) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µs is restored lead to µs-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (CH1) from µs. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem

Molecular Evaluation of Endoplasmic Reticulum Homeostasis Meets Humoral Immunity

The biosynthesis of about one third of the human proteome, including membrane receptors and secreted proteins, occurs in the endoplasmic reticulum (ER). Conditions that perturb ER homeostasis activate the unfolded protein response (UPR). An ‘optimistic’ UPR output aims at restoring homeostasis by reinforcement of machineries that guarantee efficiency and fidelity of protein biogenesis in the ER. Yet, once the UPR ‘deems’ that ER homeostatic readjustment fails, it transitions to a ‘pessimistic’ output, which, depending on the cell type, will result in apoptosis. In this article, we discuss emerging concepts on how the UPR ‘evaluates’ ER stress, how the UPR is repurposed, in particular in B cells, and how UPR-driven counter-selection of cells undergoing homeostatic failure serves organismal homeostasis and humoral immunity

Apo-parvalbumin as an Intrinsically Disordered Protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (α- and β-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in α-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge–charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-α-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20°C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10–15°C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (α-PA) or two (β-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data. Proteins 2008. © 2008 Wiley-Liss, Inc

Metal-controlled Interdomain Cooperativity in Parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca2+ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg2+- and Na+-loaded forms of pike α-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg2+- and Na+-loaded forms of pike α-PA), to the more complex mechanisms (Ca2+-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca2+-loaded pike α-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike α-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na+-bound state), thermophilic (Mg2+-form), or even of the hyperthermophilic origin (Ca2+-form)

Conversion of Human α-lactalbumin to an Apo-like State in The Complexes with Basic Poly-amino Acids: Toward Understanding of The Molecular Mechanism of Antitumor Action of HAMLET

It was recently shown that α-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem.2003, 278, 42131). In previous work, we revealed that monomeric α-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino acids (poly-Lys and poly-Arg) that represent simple models of histone proteins (Biochemistry2004, 43, 5575). Association of α-lactalbumin with histone or poly-Lys(Arg) essentially changes its properties. In the present work, the character of the changes in structural properties and conformational stability of α-lactalbumin in the complex with poly-Lys(Arg) has been studied in detail by steady-state fluorescence, circular dichroism, and differential scanning calorimetry. Complex formation strongly depends on ionic strength, confirming its electrostatic nature. Experiments with the poly-amino acids of various molecular masses demonstrated a direct proportionality between the number of α-lactalbumin molecules bound per poly-Lys(Arg) and the surface area of the poly-amino acid random coil. The binding of the poly-amino acids to Ca2+-saturated human α-lactalbumin decreases its thermal stability down to the level of its free apo-form and decreases Ca2+-affinity by 4 orders of magnitude. The conformational state of α-lactalbumin in a complex with poly-Lys(Arg), named α-LActalbumin Modified by Poly-Amino acid (LAMPA), differs from all other α-lactalbumin states characterized to date, representing an apo-like (molten globule-like) state with substantially decreased affinity for calcium ion. The requirement for efficient conversion of α-lactalbumin to the LAMPA state is a poly-Lys(Arg) chain consisting of several tens of amino acid residues

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

International audienceWe report the site-specific and covalent bioconjugation of fluorescent polymer chains to proteins in live cells using the HaloTag technology. Polymer chains bearing a Halo-ligand precisely located at their α-chain-end were synthesized in a controlled manner owing to the RAFT polymerization process. They were labeled in lateral position by several organic fluorophores such as AlexaFluor 647. The resulting Haloligand polymer probe was finally shown to selectively recognize and label HaloTag proteins present at the membrane of live cells using confocal fluorescence microscopy. Such a polymer bioconjugation approach holds great promises for various applications ranging from cell imaging to cell surface functionalization

Recoverin as a Redox-sensitive Protein

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman\u27s test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells