Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of ⍺-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin, and purified vasohibins removed the C-terminal tyrosine of ⍺-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gβγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression, protein-protein associations, or genetic interaction profiles, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.

Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, ‘FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility.

The family of integrin transmembrane receptors is essential for the normal function of multicellular organisms by facilitating cellextracellular matrix adhesion. The vitronectin-binding integrin αVβ5 localizes to focal adhesions (FAs) as well as poorly characterized flat clathrin lattices (FCLs). Here, we show that, in human keratinocytes, αVβ5 is predominantly found in FCLs, and formation of the αVβ5containing FCLs requires the presence of vitronectin as ligand, Ca 2+ , and the clathrin adaptor proteins ARH (also known as LDLRAP1), Numb and EPS15/EPS15L1. Integrin chimeras, containing the extracellular and transmembrane domains of β5 and the cytoplasmic domains of β1 or β3, almost exclusively localize in FAs. Interestingly, lowering actomyosin-mediated contractility promotes integrin redistribution to FLCs in an integrin tail-dependent manner, while increasing cellular tension favors αVβ5 clustering in FAs. Our findings strongly indicate that clustering of integrin αVβ5 in FCLs is dictated by the β5 subunit cytoplasmic domain, cellular tension and recruitment of specific adaptor proteins to the β5 subunit cytoplasmic domains.

One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a simple yet universal high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method to separate molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid profiling using full scan mass spectrometry, as well as lipid class specific scanning in positive and negative ionisation mode. This allows high-throughput processing of samples for lipidomics, even if different types of MS analysis are required. Excellent separation of isobaric and even isomeric species is achieved, and original levels of lyso-lipids can be determined without interference from lyso-lipids formed from diacyl species by source fragmentation. As examples of application of this method, more than 400 phospholipid species were identified and quantified in crude phospholipid extracts from rat liver and the parasitic helminth Schistosoma mansoni.

SummaryThe therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established a large collection of patient-derived xenografts (PDXs), derived from BRAFV600E, NRASQ61, or BRAFWT/NRASWT melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. Taking advantage of PDXs as a limitless source, we screened tumor lysates for resistance mechanisms. We identified a BRAFV600E protein harboring a kinase domain duplication (BRAFV600E/DK) in ∼10% of the cases, both in PDXs and in an independent patient cohort. While BRAFV600E/DK depletion restored sensitivity to BRAF inhibition, a pan-RAF dimerization inhibitor effectively eliminated BRAFV600E/DK-expressing cells. These results illustrate the utility of this PDX platform and warrant clinical validation of BRAF dimerization inhibitors for this group of melanoma patients.

However, these direct methods experience a high false-positive rate because of contamination by uncontrolled platelet lysis. Here, we take a reverse approach, monitoring quantitatively the concentration changes of all proteins in the platelets after platelet stimulation, whereby we assume that the released protein content should be detectable in the reduction of its level from the whole platelet proteome. For accuracy, our strategy uses stable isotope-labeling ( Figure 1A) to discriminate the released proteins from uncontrolled lysis products. In other words, most platelet proteins do not change except the ones that are significantly released on activation. Materials and MethodsMaterials and Methods are available in the online-only Supplement. 10-13 ResultsPerforming the reversed releasate quantitative proteomics experiments on 3 individuals resulted in the identification of 4375 unique proteins, of which 2970 (68%) could be quantified in ≥2 of 3 individuals (Table I in the online-only Data Supplement). Identity of the Platelet ReleasateFirst, we set out to identify the releasate on full platelet activation. The releasate was evaluated in each individual by the determination of the dimethyl intensity ratio between the light (resting) and intermediate (activated) labeled peptides ( Figure 1A). As expected, the vast majority of © 2013 American Heart Association, Inc. Objective-Platelet activation and subsequent protein release play an important role in healthy hemostasis and inflammatory responses, yet the identity and quantity of proteins in the platelet releasate are still debated. Here, we present a reversed releasate proteomics approach to determine unambiguously and quantitatively proteins released from activated platelets. Approach and Results-Isolated platelets were mock and fully stimulated after which the released proteins in the supernatant were removed. Using high-end proteomics technology (2D chromatography, stable isotope labeling, electron transfer dissociation, and high collision dissociation fragmentation) allowed us to quantitatively discriminate the released proteins from uncontrolled lysis products. Monitoring the copy numbers of ≈4500 platelet proteins, we observed that after stimulation via thrombin and collagen, only 124 (<3%) proteins were significantly released (P<0.05). The released proteins span a concentration range of ≥5 orders, as confirmed by ELISA. The released proteins were highly enriched in secretion tags and contained all known factors at high concentrations (>100 ng/mL, eg, thrombospondin, von Willebrand factor, and platelet factor 4). Interestingly, in the lower concentration range of the releasate many novel factors were identified. proteins (>95%) had a resting/activated ratio close to 1.0. These ratios were highly reproducible between replicates with a median RSD between ratios of 9.6% (Table I and Figure I in the online-only Data Supplement). Using the statistical criteria described in the Methods section in the online-only Data Supplement, 124 proteins were observed to be ...

Androgen receptor (AR) is a key player in prostate cancer development and progression. Here we applied immunoprecipitation mass spectrometry of endogenous AR in LNCaP cells to identify components of the AR transcriptional complex. In total, 66 known and novel AR interactors were identified in the presence of synthetic androgen, most of which were critical for AR-driven prostate cancer cell proliferation. A subset of AR interactors required for LNCaP proliferation were profiled using chromatin immunoprecipitation assays followed by sequencing, identifying distinct genomic subcomplexes of AR interaction partners. Interestingly, three major subgroups of genomic subcomplexes were identified, where selective gain of function for AR genomic action in tumorigenesis was found, dictated by FOXA1 and HOXB13. In summary, by combining proteomic and genomic approaches we reveal subclasses of AR transcriptional complexes, differentiating normal AR behavior from the oncogenic state. In this process, the expression of AR interactors has key roles by reprogramming the AR cistrome and interactome in a genomic location-specific manner.