Functional implications of MIR domains in protein O-mannosylation.

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a ß-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general ß-trefoil carbohydrate-binding sites (α, ß), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous ß-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.

Daily Stress, Hearing-Specific Stress and Coping: Self-reports from Deaf or Hard of Hearing Children and Children With Auditory Processing Disorder.

This study evaluated stressors and coping strategies in 70 children who are deaf or hard of hearing (D/HH) or with auditory processing disorder (APD) attending Grades 5 and 6 of a school for deaf and hard-of-hearing children. Everyday general stressors and more hearing-specific stressors were examined in a hearing-specific modified stress and coping questionnaire. Reports were compared with normative data for hearing children. Regarding everyday general stressors, stress levels for children who are D/HH or with APD did not differ from those of hearing children. Within children with hearing problems, everyday stressors were experienced as more stressful than hearing-specific stressors. For coping strategies, differences between children with hearing problems (D/HH, APD) and hearing children were shown (i.e., problem solving, anger-related emotion regulation). Girls scored higher in seeking social support whereas boys reported higher amounts of media use as a way of coping. Differences regarding stress and coping between children who are D/HH and children with APD were minor; D/HH children reported more social support seeking. Implications for assessment and resource promotion are discussed.

A conserved acidic motif is crucial for enzymatic activity of protein O-mannosyltransferases.

Protein O-mannosylation is an essential modification in fungi and mammals. It is initiated at the endoplasmic reticulum by a conserved family of dolichyl phosphate mannose-dependent protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loops 1 and 5) facing the endoplasmic reticulum lumen. Formation of dimeric PMT complexes is crucial for mannosyltransferase activity, but the direct cause is not known to date. In bakers' yeast, O-mannosylation is catalyzed largely by heterodimeric Pmt1p-Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p-Pmt2p complexes, we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially cross-linked to Pmt1p, and deletion of the loop 1 (but not loop 5) region abolished this interaction. Analysis of Pmt1p loop 1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to an Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p-Pmt2p complexes. On the basis of our data, we propose that the loop 1 regions of dimeric complexes form part of the catalytic site.

Methods to study stromal-cell derived factor 2 in the context of ER stress and the unfolded protein response in Arabidopsis thaliana.

The accumulation of misfolded or unfolded polypeptides in the endoplasmic reticulum (ER) provokes ER stress and triggers protective signaling pathways termed the unfolded protein response (UPR). Stromal cell-derived factor 2 (SDF2)-type proteins are conserved throughout the animal and plant kingdoms. Upon UPR activation transcription of SDF2-type genes is significantly enhanced in metazoan and plants, suggesting an evolutionarily conserved role. However, the precise molecular function of SDF2-type proteins still needs to be established. Most eukaryotes have two SDF2 homologous, whereas the model plant Arabidopsis thaliana has a single SDF2, thus representing an ideal model system to study the functional role of SDF2-type proteins. This chapter provides techniques to study SDF2 in the context of ER stress in Arabidopsis. We describe available sdf2 mutants, and methods to evaluate ER stress sensitivity of seedlings. Further, we summarize tools and methods that are helpful to monitor UPR induction in general (e.g., SDF2 promoter-reporter fusion constructs and SDF2-specific antibodies). In Section 6, we provide protocols for the expression and purification of recombinant SDF2 protein that can be used for further biochemical studies.

Functional and genomic analyses of blocked protein O-mannosylation in baker's yeast.

O-mannosylation is a crucial protein modification in eukaryotes that is initiated by the essential family of protein O-mannosyltransferases (PMTs). Here we demonstrate that in the model yeast Saccharomyces cerevisiae rhodanine-3-acetic acid derivatives affect members of all PMT subfamilies. Specifically, we used OGT2468 to analyse genome-wide transcriptional changes in response to general inhibition of O-mannosylation in baker's yeast. PMT inhibition results in the activation of the cell wall integrity (CWI) pathway. Coinciding, the mitogen-activated kinase Slt2p is activated in vivo and CWI pathway mutants are hypersensitive towards OGT2468. Further, induction of many target genes of the unfolded protein response (UPR) and ER-associated protein degradation (ERAD) is observed. The interdependence of O-mannosylation and UPR/ERAD is confirmed by genetic interactions between HAC1 and PMTs, and increased degradation of the ERAD substrate Pdr5p* in pmtΔ mutants. Transcriptome analyses further suggested that mating and filamentous growth are repressed upon PMT inhibition. Accordingly, in vivo mating efficiency and invasive growth are considerably decreased upon OGT2468 treatment. Quantitative PCR and ChIP analyses suggest that downregulation of mating genes is dependent on the transcription factor Ste12p. Finally, inhibitor studies identified a role of the Ste12p-dependent vegetative signalling cascade in the adaptive response to inhibition of O-mannosylation.

Arabidopsis stromal-derived Factor2 (SDF2) is a crucial target of the unfolded protein response in the endoplasmic reticulum.

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.

Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thaliana.

The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 A resolution using a synchrotron-radiation source and belonged to the hexagonal space group P6(1), with unit-cell parameters a = b = 96.1, c = 69.3 A.