Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation.

Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.

Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars.

Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC-MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease.

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1ß, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings.

Stress vulnerability shapes disruption of motor cortical neuroplasticity.

Chronic stress is a major cause of neuropsychiatric conditions such as depression. Stress vulnerability varies individually in mice and humans, measured by behavioral changes. In contrast to affective symptoms, motor retardation as a consequence of stress is not well understood. We repeatedly imaged dendritic spines of the motor cortex in Thy1-GFP M mice before and after chronic social defeat stress. Susceptible and resilient phenotypes were discriminated by symptom load and their motor learning abilities were assessed by a gross and fine motor task. Stress phenotypes presented individual short- and long-term changes in the hypothalamic-pituitary-adrenal axis as well as distinct patterns of altered motor learning. Importantly, stress was generally accompanied by a marked reduction of spine density in the motor cortex and spine dynamics depended on the stress phenotype. We found astrogliosis and altered microglia morphology along with increased microglia-neuron interaction in the motor cortex of susceptible mice. In cerebrospinal fluid, proteomic fingerprints link the behavioral changes and structural alterations in the brain to neurodegenerative disorders and dysregulated synaptic homeostasis. Our work emphasizes the importance of synaptic integrity and the risk of neurodegeneration within depression as a threat to brain health.

Respiratory status determines the effect of emodin on cell viability.

The anthraquinone emodin has been shown to have antineoplastic properties and a wealth of unconnected effects have been linked to its use, most of which are likely secondary outcomes of the drug treatment. The primary activity of emodin on cells has remained unknown. In the present study we demonstrate dramatic and extensive effects of emodin on the redox state of cells and on mitochondrial homeostasis, irrespectively of the cell type and organism, ranging from the yeast Saccharomyces cerevisiae to human cell lines and primary cells. Emodin binds to redox-active enzymes and its effectiveness depends on the oxidative and respiratory status of cells. We show that cells with efficient respiratory metabolism are less susceptible to emodin, whereas cells under glycolytic metabolism are more vulnerable to the compound. Our findings indicate that emodin acts in a similar way as known uncouplers of the mitochondrial electron transport chain and causes oxidative stress that particularly disturbs cancer cells.

Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.