Interaction interface in the C-terminal parts of centriole proteins Sas6 and Ana2

The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled–coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 in vitro and in vivo. Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2–Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously

Insight into the Structural Dynamics of the Lysenin During Prepore-to-Pore Transition Using Hydrogen–Deuterium Exchange Mass Spectrometry

Lysenin is a pore-forming toxin of the aerolysin family, which is derived from coelomic fluid of the earthworm Eisenia fetida. Upon binding to sphingomyelin (SM)-containing membranes, lysenin undergoes a series of structural changes promoting the conversion of water-soluble monomers into oligomers, leading to its insertion into the membrane and the formation of a lytic β-barrel pore. The soluble monomer and transmembrane pore structures were recently described, but the underlying structural details of oligomerization remain unclear. To investigate the molecular mechanisms controlling the conformational rearrangements accompanying pore formation, we compared the hydrogen–deuterium exchange pattern between lyseninWT and its mutant lyseninV88C/Y131C. This mutation arrests lysenin oligomers in the prepore state at the membrane surface and does not affect the structural dynamics of the water-soluble form of lysenin. In contrast, membrane-bound lyseninV88C/Y131C exhibited increased structural stabilization, especially within the twisted β-sheet of the N-terminal domain. We demonstrated that the structural stabilization of the lysenin prepore started at the site of lysenin’s initial interaction with the lipid membrane and was transmitted to the twisted β-sheet of the N-terminal domain, and that lyseninV88C/Y131C was arrested in this conformation. In lyseninWT, stabilization of these regions drove the conformational changes necessary for pore formation

A Strong Neutrophil Elastase Proteolytic Fingerprint Marks the Carcinoma Tumor Proteome

Proteolytic cascades are deeply involved in critical stages of cancer progression. During the course of peptide-wise analysis of shotgun proteomic data sets representative of colon adenocarcinoma (AC) and ulcerative colitis (UC), we detected a cancer-specific proteolytic fingerprint com- posed of a set of numerous protein fragments cleaved C-terminally to V, I, A, T, or C residues, significantly over-represented in AC. A peptide set linked by a common VIATC cleavage consensus was the only prominent can- cer-specific proteolytic fingerprint detected. This se- quence consensus indicated neutrophil elastase as a source of the fingerprint. We also found that a large frac- tion of affected proteins are RNA processing proteins associated with the nuclear fraction and mostly cleaved within their functionally important RNA-binding domains. Thus, we detected a new class of cancer-specific pep- tides that are possible markers of tumor-infiltrating neu- trophil activity, which often correlates with the clinical outcome. Data are available via ProteomeXchange with identifiers: PXD005274 (Data set 1) and PXD004249 (Data set 2). Our results indicate the value of peptide-wise anal- ysis of large global proteomic analysis data sets as op- posed to protein-wise analysis, in which outlier differen- tial peptides are usually neglected

Nucleolar Enrichment of Brain Proteins with Critical Roles in Human Neurodevelopment*

To study nucleolar involvement in brain development, the nuclear and nucleolar proteomes from the rat cerebral cortex at postnatal day 7 were analyzed using LC-MS/iTRAQ methodology. Data of the analysis are available via ProteomeXchange with identifier PXD002188. Among 504 candidate nucleolar proteins, the overrepresented gene ontology terms included such cellular compartmentcategories as “nucleolus”, “ribosome” and “chromatin”. Consistent with such classification, the most overrepresented functional gene ontology terms were related to RNA metabolism/ribosomal biogenesis, translation, and chromatin organization. Sixteen putative nucleolar proteins were associated with neurodevelopmental phenotypes in humans. Microcephaly and/or cognitive impairment were the most common phenotypic manifestations. Although several such proteins have links to ribosomal biogenesis and/or genomic stability/chromatin structure (e.g. EMG1, RPL10, DKC1, EIF4A3, FLNA, SMC1, ATRX, MCM4, NSD1, LMNA, or CUL4B), others including ADAR, LARP7, GTF2I, or TCF4 have no such connections known. Although neither the Alazami syndrome-associated LARP7nor the Pitt-Hopkins syndrome-associated TCF4 were reported in nucleoli of non-neural cells, in neurons, their nucleolar localization was confirmed by immunostaining. In cultured rat hippocampal neurons, knockdown of LARP7 reduced both perikaryal ribosome content and general protein synthesis. Similar anti-ribosomal/anti-translation effects were observed after knockdown of the ribosomal biogenesis factor EMG1 whose deficiency underlies Bowen-Conradi syndrome. Finally, moderate reduction of ribosome content and general protein synthesis followed overexpression of two Pitt-Hopkins syndrome mutant variants of TCF4. Therefore, dysregulation of ribosomal biogenesis and/or other functions of the nucleolus may disrupt neurodevelopment resulting in such phenotypes as microcephaly and/or cognitive impairment

Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis.

Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K's coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins.C.M. holds a Cambridge Cancer Centre PhD studentship, which is supported by Cancer Research UK and the MRC. Z.I.B. was supported by a Gwynaeth Pretty PhD studentship and the BlueGnome Molecular Genetics Fund from the Department of Pathology, University of Cambridge, and was also a recipient of a short-term EMBO fellowship and a fellowship from the Cambridge Philosophical Society. G.C. and M.G. are supported by a grant ‘Progetto di Ricerca di Interesse Nazionale’ (PRIN 2012) from the Ministero dell'Istruzione, Università e Ricerca. P.P.D. is the recipient of the Maplethorpe Fellowship from Murray Edwards College, Cambridge, UK.This is the final version of the article. It first appeared from Royal Society Publishing via https://doi.org/10.1098/rsob.16001

Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation.

The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of worms/flies/humans). Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. Here we show that in Drosophila, Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When we over-ride the auto-destruction of Plk4, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. We now show that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region we term the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6. We discuss these findings in light of the multiple Plk4 phosphorylation sites on Ana2

Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)

Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a “DNA cloud assay”. We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP

Mycophenolic Acid Metabolites Acyl-Glucuronide and Glucoside Affect the Occurrence of Infectious Complications and Bone Marrow Dysfunction in Liver Transplant Recipients.

BACKGROUND Mycophenolic acid (MPA) prodrugs are anti-proliferative immunosuppressive agents commonly used after organ transplantation. Although they are generally well tolerated by patients, adverse effects may occur. It is postulated that MPA metabolites could also contribute to these adverse effects. MATERIAL AND METHODS The objective of this study was the assessment of concentrations of total MPA and its metabolites, phenyl glucuronide (MPAG), acyl glucuronide (AcMPAG) and glucoside (GluMPA), using liquid chromatography combined with mass spectrometry (LC/MS/MS) in two groups: kidney transplant recipients and liver transplant patients. Associations of MPA and its metabolites with adverse effects were analyzed. RESULTS The study group consisted of 211 recipients of liver or kidney transplants who received immunosuppressive therapy, including MPA prodrugs. Multivariant analysis showed a positive influence of MPA on gastroenterotoxicity in kidney transplant recipients. In liver patients, gastroenterotoxicity was associated with lower MPAG concentrations. A positive influence of AcMPAG on bacterial infections in liver transplant patients was observed. In liver transplant recipients, a positive influence of MPA and a negative influence of GluMPA levels on the PLT count were revealed. MPA and its metabolites did not influence the hemoglobin levels in both groups. There were no significant relationships among MPA, its metabolites and WBC counts. CONCLUSIONS In kidney transplant recipients, total MPA trough concentration is associated with gastroenterotoxicity and its monitoring could have important role in management of gastrointestinal complications. The quantification of AcMPAG in liver recipients receiving MPA may be helpful in avoiding bacterial infections. GluMPA seems to have a toxic effect on thrombopoiesis

A 3′ Exonuclease that Specifically Interacts with the 3′ End of Histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication