Beside gene expression and translational control, which are relatively slow, PTM of proteins represents the major level of regulation, from very fast and reversible to slow or irreversible processes. PTMs affect protein structure and act as molecular switches, which regulate the interaction of proteins with DNA, cofactors, lipids, and other proteins. In the past few years, evidence for extensive crosstalk between PTMs has accumulated. The combination of different PTMs on protein surfaces can create a "PTM code," which can be recognized by specific effectors to initiate/inhibit downstream events, only inducing/retaining a signal once the complementary incoming signals are present at the same time and place. Although MS-based proteomics has substantially improved our knowledge about PTMs, currently sensitive and dedicated analytical strategies are available only for few different types of PTM. Several recent studies focused on the combinatorial analysis of PTMs, but preferentially utilized peptide-centric bottom-up strategies might be too restricted to decipher complex PTM codes. Here, we discuss the current state of PTM crosstalk research and how proteomics may contribute to understanding PTM codes, representing the next level of complexity and one of the biggest challenges for future proteomics research.

Carbamylation (carbamoylation) of lysine residues and protein N-termini is a nonenzymatic PTM that has been related to protein ageing. In contrast to other PTM, such as phosphorylation, carbamylation can be artificially introduced during sample preparation with urea, thus affecting studies directed toward in vivo carbamylation. In aqueous solution, urea-commonly used for denaturing proteins-is in equilibrium with ammonium and isocyanate. Under alkaline conditions, the latter can react with primary amines of free N-termini and ε-amine groups of lysines to form carbamyl derivatives. Despite being a relatively slow process, which is accelerated at elevated temperatures, prolonged incubation of protein/peptide samples in urea buffers can induce undesired carbamylation, hampering not only the proteolytic digestion with trypsin and peptide identification by MS, but also interfering with stable isotope-labeling techniques such as iTRAQ, tandem mass tags, and isotope-coded protein labeling. Here, we evaluated the extent of urea-induced carbamylation under commonly used sample preparation conditions. From our results, we can deduce that carbamylation occurs in all cases involving urea, however with varying degree: e.g. carbamidomethylation in the presence of 8.0 M urea induced carbamylation of 17% of N-termini and 4% of Lys residues. Additionally, researching a recently published large-scale dataset revealed a high degree of urea-induced carbamylation in current proteomic samples.

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrixdependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.focal adhesion | actomyosin | podocyte | FSGS

Marinesco-Sjögren syndrome (MSS) features cerebellar ataxia, mental retardation, cataracts, and progressive vacuolar myopathy with peculiar myonuclear alterations. Most MSS patients carry homozygous or compound heterozygous SIL1 mutations. SIL1 is a nucleotide exchange factor for the endoplasmic reticulum resident chaperone BiP which controls a plethora of essential processes in the endoplasmic reticulum. In this study we made use of the spontaneous Sil1 mouse mutant woozy to explore pathomechanisms leading to Sil1 deficiency-related skeletal muscle pathology. We found severe, progressive myopathy characterized by alterations of the sarcoplasmic reticulum, accumulation of autophagic vacuoles, mitochondrial changes, and prominent myonuclear pathology including nuclear envelope and nuclear lamina alterations. These abnormalities were remarkably similar to the myopathy in human patients with MSS. In particular, the presence of perinuclear membranous structures which have been reported as an ultrastructural hallmark of MSS-related myopathy could be confirmed in woozy muscles. We found that these structures are derived from the nuclear envelope and nuclear lamina and associate with proliferations of the sarcoplasmic reticulum. In line with impaired function of BiP secondary to loss of its nucleotide exchange factor Sil1, we observed activation of the unfolded protein response and the endoplasmic-reticulum-associated protein degradation-pathway. Despite initiation of the autophagy-lysosomal system, autophagic clearance was found ineffective which is in agreement with the formation of autophagic vacuoles. This report identifies woozy muscle as a faithful phenocopy of the MSS myopathy. Moreover, we provide a link between two well-established disease mechanisms in skeletal muscle, dysfunction of chaperones and nuclear envelope pathology.

Highlights d Spermidine supplementation age-protects Drosophila brain mitochondria d Brain hypusination levels decay with age but are boosted by spermidine supplementation d Mitochondrial functionality is defective after genetic attenuation of hypusination d Defective hypusination compromises spermidine effects on locomotion and memory