Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import.

Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.

Vertebrate lonesome kinase modulates the hepatocyte secretome to prevent perivascular liver fibrosis and inflammation.

Vertebrate lonesome kinase (VLK) is the only known extracellular tyrosine kinase, but its physiological functions are largely unknown. We show that VLK is highly expressed in hepatocytes of neonatal mice, but downregulated during adulthood. To determine the role of VLK in liver homeostasis and regeneration, we generated mice with a hepatocyte-specific knockout of the VLK gene (Pkdcc). Cultured progenitor cells established from primary hepatocytes of Pkdcc knockout mice produced a secretome, which promoted their own proliferation in 3D spheroids and proliferation of cultured fibroblasts. In vivo, Pkdcc knockout mice developed liver steatosis with signs of inflammation and perivascular fibrosis upon aging, combined with expansion of liver progenitor cells. In response to chronic CCl4-induced liver injury, the pattern of deposited collagen was significantly altered in these mice. The liver injury marker alpha-fetoprotein (AFP) was increased in the secretome of VLK-deficient cultured progenitor cells and in liver tissues of aged or CCl4-treated knockout mice. These results support a key role for VLK and extracellular protein phosphorylation in liver homeostasis and repair through paracrine control of liver cell function and regulation of appropriate collagen deposition. This article has an associated First Person interview with the first author of the paper.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Phosphoproteomic profiling reveals a defined genetic program for osteoblastic lineage commitment of human bone marrow-derived stromal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.

Bacterial lectin BambL acts as a B cell superantigen.

B cell superantigens crosslink conserved domains of B cell receptors (BCRs) and cause dysregulated, polyclonal B cell activation irrespective of normal BCR-antigen complementarity. The cells typically succumb to activation-induced cell death, which can impede the adaptive immune response and favor infection. In the present study, we demonstrate that the fucose-binding lectin of Burkholderia ambifaria, BambL, bears functional resemblance to B cell superantigens. By engaging surface glycans, the bacterial lectin activated human peripheral blood B cells, which manifested in the surface expression of CD69, CD54 and CD86 but became increasingly cytotoxic at higher concentrations. The effects were sensitive to BCR pathway inhibitors and excess fucose, which corroborates a glycan-driven mode of action. Interactome analyses in a model cell line suggest BambL binds directly to glycans of the BCR and regulatory coreceptors. In vitro, BambL triggered BCR signaling and induced CD19 internalization and degradation. Owing to the lectin's six binding sites, we propose a BCR activation model in which BambL functions as a clustering hub for receptor glycans, modulates normal BCR regulation, and induces cell death through exhaustive activation.

Spermidine Suppresses Age-Associated Memory Impairment by Preventing Adverse Increase of Presynaptic Active Zone Size and Release.

Memories are assumed to be formed by sets of synapses changing their structural or functional performance. The efficacy of forming new memories declines with advancing age, but the synaptic changes underlying age-induced memory impairment remain poorly understood. Recently, we found spermidine feeding to specifically suppress age-dependent impairments in forming olfactory memories, providing a mean to search for synaptic changes involved in age-dependent memory impairment. Here, we show that a specific synaptic compartment, the presynaptic active zone (AZ), increases the size of its ultrastructural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired memory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem to steer towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Spermidine feeding suppresses age-dependent memory impairment by counteracting these age-dependent changes directly at the synapse.

SPATA2 promotes CYLD activity and regulates TNF-induced NF-κB signaling and cell death.

K63- and Met1-linked ubiquitylation are crucial posttranslational modifications for TNF receptor signaling. These non-degradative ubiquitylations are counteracted by deubiquitinases (DUBs), such as the enzyme CYLD, resulting in an appropriate signal strength, but the regulation of this process remains incompletely understood. Here, we describe an interaction partner of CYLD, SPATA2, which we identified by a mass spectrometry screen. We find that SPATA2 interacts via its PUB domain with CYLD, while a PUB interaction motif (PIM) of SPATA2 interacts with the PUB domain of the LUBAC component HOIP SPATA2 is required for the recruitment of CYLD to the TNF receptor signaling complex upon TNFR stimulation. Moreover, SPATA2 acts as an allosteric activator for the K63- and M1-deubiquitinase activity of CYLD In consequence, SPATA2 substantially attenuates TNF-induced NF-κB and MAPK signaling. Conversely, SPATA2 is required for TNF-induced complex II formation, caspase activation, and apoptosis. Thus, this study identifies SPATA2 as an important factor in the TNF signaling pathway with a substantial role for the effects mediated by the cytokine.

Metadherin exon 11 skipping variant enhances metastatic spread of ovarian cancer.

Metastatic ovarian cancer has a dismal prognosis and current chemotherapeutic approaches have very limited success. Metadherin (MTDH) is expressed in human ovarian cancer tissue and its expression inversely correlates with patients overall survival. Consistent with these studies, we observed MTDH expression in tissue specimens of FIGO Stage III ovarian carcinomas (72/83 cases). However, we also observed this in normal human ovarian epithelial (OE) cells, which raised the question of whether MTDH-variants with functional differences exist. We identified a novel MTDH exon 11 skipping variant (MTDHdel) which was seen at higher levels in ovarian cancer compared to benign OE cells. We analyzed MTDH-binding partner interactions and found that 12 members of the small ribosomal subunit and several mRNA binding proteins bound stronger to MTDHdel than to wildtype MTDH which indicates differential effects on gene translation. Knockdown of MTDH in ovarian cancer cells reduced the amount of distant metastases and improved the survival of ovarian cancer-bearing mice. Selective overexpression of the MTDHdel enhanced murine and human ovarian cancer progression and caused a malignant phenotype in originally benign human OE cells. MTDHdel was detectable in microdissected ovarian cancer cells of some human tissue specimens of ovarian carcinomas. In summary, we have identified a novel MTDH exon 11 skipping variant that shows enhanced binding to small ribosomal subunit members and that caused reduced overall survival of ovarian cancer bearing mice. Based on the findings in the murine system and in human tissues, MTDHdel must be considered a major promalignant factor for ovarian cancer.

RhoA activation by CNFy restores cell-cell adhesion in kindlin-2-deficient keratinocytes.

Kindlins are a family of integrin adapter and cell-matrix adhesion proteins causally linked to human genetic disorders. Kindlin-2 is a ubiquitously expressed protein with manifold functions and interactions. The contribution of kindlin-2 to integrin-based cell-matrix adhesions has been extensively explored, while other integrin-independent roles emerge. Because of the early involvement of kindlin-2 in development, no viable animal models with its constitutional knockout are available to study its physiological functions in adult skin. Here, we uncovered a critical physiological role of kindlin-2 in the epidermis by using a skin-equivalent model with shRNA-mediated knock-down of kindlin-2 in keratinocytes. Kindlin-2-deficient keratinocytes built stratified epidermal layers, but displayed impaired dermal-epidermal and intra-epidermal adhesion and barrier function. Co-immunoprecipitation studies demonstrated that kindlin-2 interacts with both integrin- and cadherin-based adhesions. In kindlin-2-deficient keratinocytes, reduced cell-cell adhesion was associated with abnormal cytoplasmic distribution of adherens junctions and desmosomal proteins, which was dependent on RhoA activation. Direct activation of RhoA with recombinant bacterial cytotoxic necrotizing factor y (CNFy) reverted the abnormal phenotype and barrier function of kindlin-2-deficient keratinocytes and skin equivalents. These findings have physiological and pathological significance, since kindlin-2 expression modulates the phenotype in Kindler syndrome, a skin fragility disorder caused by kindlin-1 deficiency. Our results suggest that pharmacological regulation of RhoGTPase activity may represent a therapeutic option for skin fragility.

The quantitative nuclear matrix proteome as a biochemical snapshot of nuclear organization.

The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining bona fide NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology.

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.

Rapid combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

Protein phosphorylation is an important mechanism of cellular signaling, and many proteins are precisely regulated through the interplay of stimulatory and inhibitory phosphorylation sites. Phosphoproteomics offers great opportunities to unravel this complex interplay, generating a mechanistic understanding of vital cellular processes. However, protein phosphorylation is substoichiometric and, in particular, peptides carrying multiple phosphorylation sites are extremely difficult to detect in a highly complex mixture of abundant nonphosphorylated peptides. Chromatographic methods are employed to reduce sample complexity and thereby significantly increase the number of phosphopeptide identifications. We previously demonstrated that combinatorial strong cation exchange-electrostatic repulsion-hydrophilic interaction chromatography yields a surplus in overall identifications of phosphopeptides compared with single chromatographic approaches. Here we present a simple and rapid strategy implemented as solid-phase extraction not requiring specific instrumentation such as off-line HPLC systems. It is inexpensive, adaptable for high and low amounts of starting material, and saves time by allowing multiplexed sample preparation without any carry-over problem.

Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks.

A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.

Collagen constitutes about 12% in females and 17% in males of the total protein in mice.

Collagen has been postulated to be the most abundant protein in our body, making up one-third of the total protein content in mammals. However, a direct assessment of the total collagen levels of an entire mammal to confirm this estimate is missing. Here we measured hydroxyproline levels as a proxy for collagen content together with total protein levels of entire mice or of individual tissues. Collagen content normalized to the total protein is approximately 0.1% in the brain and liver, 1% in the heart and kidney, 4% in the muscle and lung, 6% in the colon, 20-40% in the skin, 25-35% in bones, and 40-50% in tendons of wild-type (CD1 and CB57BL/6) mice, consistent with previous reports. To our surprise, we find that collagen is approximately 12% in females and 17% in males of the total protein content of entire wild-type (CD1 and CB57BL/6) mice. Although collagen type I is the most abundant collagen, the most abundant proteins are albumin, hemoglobulin, histones, actin, serpina, and then collagen type I. Analyzing amino acid compositions of mice revealed glycine as the most abundant amino acid. Thus, we provide reference points for collagen, matrisome, protein, and amino acid composition of healthy wild-type mice.

The inflammation repressor TNIP1/ABIN-1 is degraded by autophagy following TBK1 phosphorylation of its LIR.

The inflammatory repressor TNIP1/ABIN-1 is important for keeping in check inflammatory and cell-death pathways to avoid potentially dangerous sustained activation of these pathways. We have now found that TNIP1 is rapidly degraded by selective macroautophagy/autophagy early (0-4 h) after activation of TLR3 by poly(I:C)-treatment to allow expression of pro-inflammatory genes and proteins. A few hours later (6 h), TNIP1 levels rise again to counteract sustained inflammatory signaling. TBK1-mediated phosphorylation of a TNIP1 LIR motif regulates selective autophagy of TNIP1 by stimulating interaction with Atg8-family proteins. This is a novel level of regulation of TNIP1, whose protein level is crucial for controlling inflammatory signaling.

Profiling of purified autophagic vesicle degradome in the maturing and aging brain.

Autophagy disorders prominently affect the brain, entailing neurodevelopmental and neurodegenerative phenotypes in adolescence or aging, respectively. Synaptic and behavioral deficits are largely recapitulated in mouse models with ablation of autophagy genes in brain cells. Yet, the nature and temporal dynamics of brain autophagic substrates remain insufficiently characterized. Here, we immunopurified LC3-positive autophagic vesicles (LC3-pAVs) from the mouse brain and proteomically profiled their content. Moreover, we characterized the LC3-pAV content that accumulates after macroautophagy impairment, validating a brain autophagic degradome. We reveal selective pathways for aggrephagy, mitophagy, and ER-phagy via selective autophagy receptors, and the turnover of numerous synaptic substrates, under basal conditions. To gain insight into the temporal dynamics of autophagic protein turnover, we quantitatively compared adolescent, adult, and aged brains, revealing critical periods of enhanced mitophagy or degradation of synaptic substrates. Overall, this resource unbiasedly characterizes the contribution of autophagy to proteostasis in the maturing, adult, and aged brain.

Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis.

Macroautophagy/autophagy is an evolutionarily conserved cellular stress response mechanism. Autophagy induction in the tumor microenvironment (stroma) has been shown to support tumor metabolism. However, cancer cell-derived secreted factors that initiate communication with surrounding cells and stimulate autophagy in the tumor microenvironment are not fully documented. We identified CTF1/CT-1 (cardiotrophin 1) as an activator of autophagy in fibroblasts and breast cancer-derived carcinoma-associated fibroblasts (CAFs). We showed that CTF1 stimulated phosphorylation and nuclear translocation of STAT3, initiating transcriptional activation of key autophagy proteins. Additionally, following CTF1 treatment, AMPK and ULK1 activation was observed. We provided evidence that autophagy was important for CTF1-dependent ACTA2/α-SMA accumulation, stress fiber formation and fibroblast activation. Moreover, promotion of breast cancer cell migration and invasion by activated fibroblasts depended on CTF1 and autophagy. Analysis of the expression levels of CTF1 in patient-derived breast cancer samples led us to establish a correlation between CTF1 expression and autophagy in the tumor stroma. In line with our in vitro data on cancer migration and invasion, higher levels of CTF1 expression in breast tumors was significantly associated with lymph node metastasis in patients. Therefore, CTF1 is an important mediator of tumor-stroma interactions, fibroblast activation and cancer metastasis, and autophagy plays a key role in all these cancer-related events.Abbreviations: ACTA2/α-SMA: actin, alpha 2, smooth muscle CAFs: cancer- or carcinoma-associated fibroblasts CNT Ab.: control antibody CNTF: ciliary neurotrophic factor CTF1: cardiotrophin 1 CTF1 Neut. Ab.: CTF1-specific neutralizing antibody GFP-LC3 MEF: GFP-fused to MAP1LC3 protein transgenic MEF LIF: leukemia inhibitory factor IL6: interleukin 6 MEFs: mouse embryonic fibroblasts MEF-WT: wild-type MEFs OSM: oncostatin M TGFB/TGFß: transforming growth factor beta.

Combinatorial use of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and strong cation exchange (SCX) chromatography for in-depth phosphoproteome analysis.

In large-scale phosphoproteomics studies, fractionation by strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is commonly used to reduce sample complexity, fractionate phosphopeptides from their unmodified counterparts, and increase the dynamic range for phosphopeptide identification. However, these procedures do not succeed to separate, both singly and multiply phosphorylated peptides due to their inverse physicochemical characteristics. Hence, depending on the chosen method only one of the two peptide classes can be efficiently separated. Here, we present a novel strategy based on the combinatorial separation of singly and multiply phosphorylated peptides by SCX and ERLIC for in-depth phosphoproteome analysis. In SCX, mostly singly phosphorylated peptides are retained and fractionated while not-retained multiply phosphorylated peptides are fractionated in a subsequent ERLIC approach (SCX-ERLIC). In ERLIC, multiply phosphorylated peptides are fractionated, while not-retained singly phosphorylated peptides are separated by SCX (ERLIC-SCX). Compared to single step fractionations by SCX, the combinatorial strategies, SCX-ERLIC and ERLIC-SCX, yield up to 48% more phosphopeptide identifications as well as a strong increase in the number of detected multiphosphorylated peptides. Phosphopeptides identified in two subsequent, complementary fractionations had little overlap (5%) indicating that ERLIC and SCX are orthogonal methods ideally suited for in-depth phosphoproteome studies.