Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively

Cleavable Crosslinkers as Tissue Fixation Reagents for Proteomic Analysis

Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced crosslinks, fixed tissue proteins are difficult to extract, which hampers mass spectrometry (MS) proteomic analyses. Recent years have seen the use of different combinations of high temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixed paraffin-embedded tissue proteomes. However, to achieve protein extraction yields similar to those of fresh-frozen tissue, high-temperature heating is necessary. Such harsh extraction conditions can affect sensitive amino acids and post-translational modifications, resulting in the loss of important information, while still not resulting in protein yields comparable to those of fresh-frozen tissue. Herein, the objective is to evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation and efficient protein extraction from fixed tissue for MS proteomics under mild conditions. With this goal in mind, disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) are investigated as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups, leading to amide bond formation, and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP), respectively. Results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to that of formaldehyde. In addition, cleavage of DSP improves protein recovery from fixed tissue by a factor of 18 and increases the number of identified proteins by approximately 20 % under mild extraction conditions compared with those of formaldehyde-fixed paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, although effective, results in side reactions that prevent effective protein extraction and interfere with protein identification. Protein crosslinkers that can be cleaved under mild conditions and result in defined modifications, such as DSP, are thus viable alternatives to formaldehyde as tissue fixatives to facilitate protein analysis from paraffin-embedded, fixed tissue

A murine model of Charcot-Marie-Tooth disease 4F reveals a role for the C-terminus of periaxin in the formation and stabilization of Cajal bands

Charcot-Marie-Tooth (CMT) disease comprises up to 80 monogenic inherited neuropathies of the peripheral nervous system (PNS) that collectively result in demyelination and axon degeneration. The majority of CMT disease is primarily either dysmyelinating or demyelinating in which mutations affect the ability of Schwann cells to either assemble or stabilize peripheral nerve myelin. CMT4F is a recessive demyelinating form of the disease caused by mutations in the Periaxin (PRX) gene. Periaxin (Prx) interacts with Dystrophin Related Protein 2 (Drp2) in an adhesion complex with the laminin receptor Dystroglycan (Dag). In mice the Prx/Drp2/Dag complex assembles adhesive domains at the interface between the abaxonal surface of the myelin sheath and the cytoplasmic surface of the Schwann cell plasma membrane. Assembly of these appositions causes the formation of cytoplasmic channels called Cajal bands beneath the surface of the Schwann cell plasma membrane. Loss of either Periaxin or Drp2 disrupts the appositions and causes CMT in both mouse and man. In a mouse model of CMT4F, complete loss of Periaxin first prevents normal Schwann cell elongation resulting in abnormally short internodal distances which can reduce nerve conduction velocity, and subsequently precipitates demyelination. Distinct functional domains responsible for Periaxin homodimerization and interaction with Drp2 to form the Prx/Drp2/Dag complex have been identified at the N-terminus of Periaxin. However, CMT4F can also be caused by a mutation that results in the truncation of Periaxin at the extreme C-terminus with the loss of 391 amino acids. By modelling this in mice, we show that loss of the C-terminus of Periaxin results in a surprising reduction in Drp2. This would be predicted to cause the observed instability of both appositions and myelin, and contribute significantly to the clinical phenotype in CMT4F

Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis

Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

AbstractThe temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.</jats:p

Wandel gestalten. Aufgaben und Randbedingungen des (digitalen) Publizierens heute

Aus der Sicht eines Programmleiters wird dargestellt, was die Aufgabe verlegerischen Tuns war und heute noch ist und welche Herausforderungen sich heute für Verlage stellen. Exemplarisch wird das am VS Verlag für Sozialwissenschaften demonstriert. (DIPF/Mar.