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

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

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

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen

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

3D model of EDEM1 and sequence alignment to ER class I α1–2 mannosidase template.

<p>A. Refined structural alignment of EDEM1 to ER class I α1–2 mannosidase. <i>EDEM_cDis</i>; _<i>cSS</i> & _<i>rms</i> are the <i>consensus disorder</i>, <i>consensus secondary structure</i> and <i>model root mean deviation</i> from an optimal alpha carbon path, in Angstroms, respectively. B. The 3D model of EDEM1. The ID region is shown in dark-red. Helices are shown in red and beta structures in yellow. N-glycans are shown in green lines. A potential Man5 substrate is shown in orange. The calcium ion and the three main aminoacids assessed to be involved in substrate binding and processing are shown in magenta.</p

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

Cloning of EDEM1 mutants and their half-lifes and localization.

<p>A. Schematic representation of EDEM1 mutants. The putative transmembrane domain is oval, the mannosidase-like domain (MLD) is, the deletion of the ID region and the point mutants are shown in red. B. EDEM1, EDEM1- ΔIDR and EDEM1-AVV were expressed in HEK 293T cells. The cells were pulse labeled with [³⁵S]-Methionine/Cysteine for 20 min and chased for 0 min. Half of the cell lysates were immunoprecipitated with anti-HA antibodies and the other half with anti-EDEM1 antibodies and analyzed by SDS-PAGE followed by autoradiography. Arrowhead indicates EDEM1 band, horizontal line highlights the two bands of EDEM1-ΔIDR and the asterisk points out EDEM1-AVV band. C. 293T cells overexpressing the three EDEM1 proteins grown in 3,5 cm culture dishes were lysed and one tenth of the cell lysate was loaded on gel separated by SDS-PAGE and blotted with goat anti-HA or rabbit anti-EDEM1 antibodies. D. Cells expressing EDEM1 or EDEM2 recombinant constructs were used for western blot experiments, similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042998#pone-0042998-g002" target="_blank">Figure 2C</a>, using rabbit α-EDEM1 or rabbit α-EDEM2 antibodies as mentioned in the figure. E. EDEM1 mutants were transfected in HEK 293T cells, pulse-labeled for 30 min and chased for 0 min. Cell lysates were immunoprecipitated with rabbit anti-EDEM1 antibodies. Samples were divided in two an subjected to EndoH digestion over night and solved by SDS-PAGE. F. To determine the rate of degradation of EDEM1 mutants, HEK 293T cells overexpressing the EDEM mutants were labeled with [³⁵S]-Methionine/Cysteine for 20 min and chased for 0 up to 6 h. Total lysates were immunoprecipitated with anti-EDEM1 antiserum and the isolated proteins were separated by SDS-PAGE and visualized by autoradiography. G. The graph shows the average percent EDEM1 remaining after chase relative to corresponding pulse, using Image J software, mean of three independent experiments. H. A375 cells were grown on coverslips for one day, transfected with cDNA encoding for the three EDEM1 mutants and processed for immunofluorescence with polyclonal anti-EDEM1 and rabbit anti-calnexin antibodies.</p