48 research outputs found
Interface Analysis of the Complex between ERK2 and PTP-SL
The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2–PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex
A Caenorhabditis elegans Wild Type Defies the Temperature–Size Rule Owing to a Single Nucleotide Polymorphism in tra-3
Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature–size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature–size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature–size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 × CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature–size rule, which has puzzled biologists for decades
C-Terminus Glycans with Critical Functional Role in the Maturation of Secretory Glycoproteins
The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I
Harmonicity in slow protein dynamics
International audienceThe slow dynamics of proteins around its native folded state is usually described by diffusion in a strongly anharmonic potential. In this paper, we try to understand the form and origin of the anharmonicities, with the principal aim of gaining a better understanding of the principal motion types, but also in order to develop more efficient numerical methods for simulating neutron scattering spectra of large proteins. First, we decompose a molecular dynamics (MD) trajectory of 1.5 ns for a C-phycocyanin dimer surrounded by a layer of water into three contributions that we expect to be independent: the global motion of the residues, the rigid-body motion of the sidechains relative to the backbone, and the internal deformations of the sidechains. We show that they are indeed almost independent by verifying the factorization of the incoherent intermediate scattering function. Then, we show that the global residue motions, which include all large-scale backbone motions, can be reproduced by a simple harmonic model which contains two contributions: a short-time vibrational term, described by a standard normal mode calculation in a local minimum, and a long-time diffusive term, described by Brownian motion in an effective harmonic potential. The potential and the friction constants were fitted to the MD data. The major anharmonic contribution to the incoherent intermediate scattering function comes from the rigid-body diffusion of the sidechains. This model can be used to calculate scattering functions for large proteins and for long-time scales very efficiently, and thus provides a useful complement to MD simulations, which are best suited for detailed studies on smaller systems or for shorter time scales
Tyrosinase Degradation Is Prevented when EDEM1 Lacks the Intrinsically Disordered Region
<div><p>EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121–598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40–119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.</p> </div
Methionine oxidation selectively enhances T cell reactivity against a melanoma antigen
Summary: The impact of the peptide amino acids side-chain modifications on the immunological recognition has been scarcely explored. We investigate here the effect of methionine oxidation on the antigenicity of the melanoma immunodominant peptide 369-YMDGTMSQV-377 (YMD). Using CD8+ T cell activation assays, we found that the antigenicity of the sulfoxide form is higher when compared to the YMD peptide. This is consistent with free energy computations performed on HLA-A∗02:01/YMD/TCR complex showing that this is lowered upon oxidation, paired with a steep increase in order at atomic level. Oxidized YMD forms were identified at the melanoma cell surface by LC-MS/MS analysis. These results demonstrate that methionine oxidation in the antigenic peptides may generate altered peptide ligands with increased antigenicity, and that this oxidation may occur in vivo, opening up the possibility that high-affinity CD8+ T cells might be naturally primed in the course of melanoma progression, as a result of immunosurveillance
Tyrosinase Degradation Is Prevented when EDEM1 Lacks the Intrinsically Disordered Region
Association of EDEM1 mutants with calnexin and SEL1L.
<p>A. HEK 293T cells overexpressing EDEM1 mutants were radioactively labeled for 30 min, chased for 0 and 15 min, harvested and lysed. Cells were immunoprecipitated with anti-EDEM1 or anti-calnexin antibodies and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042998#pone-0042998-g004" target="_blank">Figure 4</a>. B. (Upper panel). Cells expressing EDEM1 mutants were pulse-labeled for 20 min and chased for 1 h. Half of the cellular lysates were used for immunoprecipitation with anti-SEL1L antibodies and the other half was used for immunoprecipitation with anti-EDEM1 antiserum and analyzed as in A. EDEM1 mutants co-precipitate with SEL1L protein. (Middle panel). Cells expressing EDEM1 mutants were immunoprecipitated with anti-EDEM1 antibodies. The immunocomplexes along with total lysates (TL) controls were separated by SDS-PAGE and Western-blotted with anti- SEL1L antibodies. (Lower panel). Total lysates from the previously described experiment were used for gel electrophoresis and Western blot with EDEM1 antibodies to test the expression of overexpressed mutants. C. A375 cells expressing EDEM1 mutants were processed for immunofluorescence, as described in Experimental procedures, with anti-SEL1L and anti-EDEM1 antibodies.</p
Tyrosinase mutants do not associate with EDEM1 in the absence of the ID region.
<p>A. 293T cells were co-transfected tyrosinase mutants, pulse labeled and chased for the indicated time points and the imunocomplexes were isolated using tyrosinase T311 or HA antibodies, separated on gel and visualized by autoradiography. B. The deletion mutant of EDEM1 (EDEM1-ΔIDR) was subjected to the same type of experiment as above, using tyrosinase and EDEM1 polyclonal antibodies. Mock samples were cells transfected with tyrosinase (mock 1) or EDEM1 (mock 2). C. EDEM1-AVV was co-expressed with tyrosinase wild type and soluble mutant and subsequently used for metabolic labeling for 20 min, harvested and immunoprecipitated with tyrosinase or EDEM1 antibodies. D. NG-TYR was co-transfected with EDEM1 mutants in 293T cells which were pulse labeled for 20 min and immunoprecipitated with EDEM1 or tyrosinase antibodies. As shown wild type and triple mutant of EDEM1 coprecipitate with non-glycosylated tyrosinase, but not the truncated EDEM1 mutant(EDEM1-ΔIDR). E. EDEM1 and tyrosinase mutants were expressed in HEK293T cells, pulse labeled 20 min and immunoprecipitated with corresponding antibodies as control for the previously discussed co-precipitations.</p