Identification of proprotein convertase substrates using genome-wide expression correlation analysis

Identification of proprotein convertase substrates using genome-wide expression correlation analysis Turpeinen, Hannu Kukkurainen, Sampo Pulkkinen, Kati Kauppila, Timo Ojala, Kalle Hytonen, Vesa P Pesu, Marko England BMC genomics BMC Genomics. 2011 Dec 20;12:618. engABSTRACT: BACKGROUND: Subtilisin/kexin-like proprotein convertase (PCSK) enzymes have important regulatory function in a wide variety of biological processes. PCSKs proteolytically process at a target sequence that contains basic amino acids arginine and lysine, which results in functional maturation of the target protein. In vitro assays have showed significant biochemical redundancy between the seven family members, but the phenotypes of PCSK deficient mice and patients carrying an inactive PCSK allele argue for a specific biological function. Modeling the structures of individual PCSK enzymes has offered little insights into the specificity determinants. However, previous studies have shown that there can be a coordinated expression between a PCSK and its target molecule. Here, we have surveyed the putative PCSK target proteins using genome-wide expression correlation analysis and cleavage site prediction algorithms. RESULTS: We first performed a gene expression correlation analysis over the whole genome for all PCSK enzymes. PCSKs were found to cluster differently based on the strength of correlations. The screen for putative PCSK target proteins showed a significant enrichment (p-values from 1.2e-4 to <1.0e-10) of putative targets among the most positively correlating genes for most PCSKs. Interestingly, there was no enrichment in putative targets among the genes that correlated positively with the biologically redundant PCSK7, whereas PCSK5 showed an inverse correlation. PCSKs also showed a highly variable degree of shared target genes that were identified by expression correlation and cleavage site prediction. Multiple alignments were used to evaluate the putative targets to pinpoint the important residues for the substrate recognition. Finally, we validated our approach and identified biochemically PAPPA1 and ADAMTS6 as novel targets for FURIN proteolytic activity. CONCLUSIONS: Most PCSK enzymes display strong positive expression correlation with predicted target proteins in our genome-wide analysis. We also show that expression correlation screen combined with a cleavage site-prediction analysis can be used to identify novel bona fide target molecules for PCSKs. Exploring the positively correlating genes can thus offer additional insights into the biology of proprotein convertases.Peer reviewe

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the beta integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate beta 3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for beta 3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of alpha- and beta 3-integrins, in order to activate the beta 3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the beta 3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer. This article has an associated First Person interview with the first author of the paper.Peer reviewe

Zebavidin - An avidin-like protein from zebrafish

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.Public Library of Science open acces

Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins

BACKGROUND: Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications. PRINCIPAL FINDINGS: To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation. SIGNIFICANCE: The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity

Taliini-integriini-vuorovaikutuksen molekyylidynamiikkaa

Solut tunnustelevat ympäristöään ja tarttuvat alustaansa integriineillä, solukalvon reseptoriproteiineilla. Taliini-proteiini sitoutuu integriinin solunsisäiseen häntään, aktivoi integriinin ja kytkee sen solutukirankaan. Taliini-integriini-kompleksi välittää mekaanisia signaaleja soluväliaineen ja solutukirangan välillä, ja taliiniin näin kohdistuva venytys kytkee päälle ja pois päältä erilaisia signaalireittejä. Nämä mekaaniset ja biokemialliset signaalit puolestaan vaikuttavat solun muotoon ja liikkumiseen sekä geenien ilmentymiseen. Taliinin ja integriinin vuorovaikutuksia on tutkittu laajasti, mutta vielä ei ole selvää, miten taliini aktivoi integriinin. Taliinin integriiniä aktivoivan rakenneosan koko rakennetta ei myöskään ole pystytty selvittämään. Väitöskirjassani keskityn tutkimaan integriinin ja taliinin vuorovaikutuksia molekyylidynamiikkasimulaatioilla. Väitöskirjan ensimmäisessä osatyössä tutkimme miten taliinin ja integriinin vuorovaikutukset muuttuvat mekaanisessa rasituksessa. Taliiniin ja integriiniin kohdistuvaa rasitusta mallinsimme vetämällä näitä irralleen toisistaan molekyylidynamiikkasimulaatioissa. Havaitsimme, että taliini-integriini-kompleksi kesti hyvin rasitusta, kun integriini oli sitoutuneena taliiniin vastakkaissuuntaisena β-säikeenä, ja integriinin NPxY-motiivi sitoutui taliiniin vain löyhästi. Toisessa ja kolmannessa osatyössä tutkimme taliinin ja αIIbβ3-integriinin vuorovaikutuksia simulaatioissa, jossa oli mukana solukalvomalli. Fosfoinositidien tiedetään osallistuvan integriinin toiminnan säätelyyn, ja sisällytimmekin osassa simulaatioista solukalvoon neutraalien fosfolipidien lisäksi negatiivisesti varautuneita fosfoinositideja. Näissä simulaatioissa tarkasteltiin sekä julkaistuun kristallografiseen rakenteeseen pohjautuvaa pitkänomaista mallia että kompaktia muotoa, jonka mallinsin taliinin sekvenssihomologian perusteella. Pitkänomaiseen muotoon perustuvissa simulaatioissa havaitsimme, että solukalvon fosfoinositidit voivat häiritä integriinin alayksiköiden välisiä vuorovaikutuksia, ja mahdollisesti myös aktivoida integriiniä. Kompaktilla taliinimallilla tehdyistä simulaatioista ennustimme, että taliinin joustava silmukkarakenne asettuu solukalvolla suoraan kontaktiin integriinin kanssa. Biofysikaaliset kokeet ja solukokeet tukevat sekä kompaktia taliinimalliamme että taliinin silmukkarakenteen ja integriinin välistä vuorovaikutusta.Cells sense their environment and adhere to substrates with the help of cell surface receptors, integrins. The activity of integrins is controlled by talin, a large intracellular adaptor protein binding to the cytoplasmic tail of integrin. Talin both activates integrin and connects it to the force-bearing machinery of the cell. The talin-integrin complex mediates mechanical signals between the extracellular matrix and the intracellular actin cytoskeleton, and the tension dictates the coupling of further adaptor and signaling proteins to talin. These mechanical and biochemical signals affect the shape and motion of the cell, and regulate gene expression. Despite numerous studies on talin-integrin interactions, it is not yet fully clear how talin activates integrin, and the complete structure of the integrin-binding talin head domain remains to be solved. In this thesis I focus on the interactions of integrin and the talin head domain using molecular dynamics simulations. In the first study we analyzed how mechanical load affects the talin-integrin binding interface in a set of constant force pulling simulations. We found that the mechanical stability of the talin-integrin interface requires a set of anti-parallel β-sheet-like hydrogen bonds, but not the binding of the β-integrin NPxY motif. In the second and third studies, we studied the talin-αIIbβ3 integrin complex in the presence of a lipid bilayer. These simulations were carried out using two different talin models: an extended talin head conformation based on a published crystallographic structure, and our proposed compact talin conformation, whose design is based on homologous 3-D structures. Simulations of the extended talin conformation showed that the integrin dimer could become perturbed, and even activated, by acidic phospholipids in the cell membrane. With our own, compact domain model, our MD simulations predicted an interaction between a flexible loop and integrin. Experiments carried out in vitro and in cellulo supported the proposed compact conformation of the talin head and provided evidence for the predicted interaction between the talin loop and integrin

Taliini-integriini-vuorovaikutuksen molekyylidynamiikkaa

Solut tunnustelevat ympäristöään ja tarttuvat alustaansa integriineillä, solukalvon reseptoriproteiineilla. Taliini-proteiini sitoutuu integriinin solunsisäiseen häntään, aktivoi integriinin ja kytkee sen solutukirankaan. Taliini-integriini-kompleksi välittää mekaanisia signaaleja soluväliaineen ja solutukirangan välillä, ja taliiniin näin kohdistuva venytys kytkee päälle ja pois päältä erilaisia signaalireittejä. Nämä mekaaniset ja biokemialliset signaalit puolestaan vaikuttavat solun muotoon ja liikkumiseen sekä geenien ilmentymiseen. Taliinin ja integriinin vuorovaikutuksia on tutkittu laajasti, mutta vielä ei ole selvää, miten taliini aktivoi integriinin. Taliinin integriiniä aktivoivan rakenneosan koko rakennetta ei myöskään ole pystytty selvittämään. Väitöskirjassani keskityn tutkimaan integriinin ja taliinin vuorovaikutuksia molekyylidynamiikkasimulaatioilla. Väitöskirjan ensimmäisessä osatyössä tutkimme miten taliinin ja integriinin vuorovaikutukset muuttuvat mekaanisessa rasituksessa. Taliiniin ja integriiniin kohdistuvaa rasitusta mallinsimme vetämällä näitä irralleen toisistaan molekyylidynamiikkasimulaatioissa. Havaitsimme, että taliini-integriini-kompleksi kesti hyvin rasitusta, kun integriini oli sitoutuneena taliiniin vastakkaissuuntaisena β-säikeenä, ja integriinin NPxY-motiivi sitoutui taliiniin vain löyhästi. Toisessa ja kolmannessa osatyössä tutkimme taliinin ja αIIbβ3-integriinin vuorovaikutuksia simulaatioissa, jossa oli mukana solukalvomalli. Fosfoinositidien tiedetään osallistuvan integriinin toiminnan säätelyyn, ja sisällytimmekin osassa simulaatioista solukalvoon neutraalien fosfolipidien lisäksi negatiivisesti varautuneita fosfoinositideja. Näissä simulaatioissa tarkasteltiin sekä julkaistuun kristallografiseen rakenteeseen pohjautuvaa pitkänomaista mallia että kompaktia muotoa, jonka mallinsin taliinin sekvenssihomologian perusteella. Pitkänomaiseen muotoon perustuvissa simulaatioissa havaitsimme, että solukalvon fosfoinositidit voivat häiritä integriinin alayksiköiden välisiä vuorovaikutuksia, ja mahdollisesti myös aktivoida integriiniä. Kompaktilla taliinimallilla tehdyistä simulaatioista ennustimme, että taliinin joustava silmukkarakenne asettuu solukalvolla suoraan kontaktiin integriinin kanssa. Biofysikaaliset kokeet ja solukokeet tukevat sekä kompaktia taliinimalliamme että taliinin silmukkarakenteen ja integriinin välistä vuorovaikutusta.Cells sense their environment and adhere to substrates with the help of cell surface receptors, integrins. The activity of integrins is controlled by talin, a large intracellular adaptor protein binding to the cytoplasmic tail of integrin. Talin both activates integrin and connects it to the force-bearing machinery of the cell. The talin-integrin complex mediates mechanical signals between the extracellular matrix and the intracellular actin cytoskeleton, and the tension dictates the coupling of further adaptor and signaling proteins to talin. These mechanical and biochemical signals affect the shape and motion of the cell, and regulate gene expression. Despite numerous studies on talin-integrin interactions, it is not yet fully clear how talin activates integrin, and the complete structure of the integrin-binding talin head domain remains to be solved. In this thesis I focus on the interactions of integrin and the talin head domain using molecular dynamics simulations. In the first study we analyzed how mechanical load affects the talin-integrin binding interface in a set of constant force pulling simulations. We found that the mechanical stability of the talin-integrin interface requires a set of anti-parallel β-sheet-like hydrogen bonds, but not the binding of the β-integrin NPxY motif. In the second and third studies, we studied the talin-αIIbβ3 integrin complex in the presence of a lipid bilayer. These simulations were carried out using two different talin models: an extended talin head conformation based on a published crystallographic structure, and our proposed compact talin conformation, whose design is based on homologous 3-D structures. Simulations of the extended talin conformation showed that the integrin dimer could become perturbed, and even activated, by acidic phospholipids in the cell membrane. With our own, compact domain model, our MD simulations predicted an interaction between a flexible loop and integrin. Experiments carried out in vitro and in cellulo supported the proposed compact conformation of the talin head and provided evidence for the predicted interaction between the talin loop and integrin

Molecular Dynamics of Talin-Integrin Interaction

Cells sense their environment and adhere to substrates with the help of cell surface receptors,integrins. The activity of integrins is controlled by talin, a large intracellular adaptor proteinbinding to the cytoplasmic tail of integrin. Talin both activates integrin and connects it to theforce-bearing machinery of the cell. The talin-integrin complex mediates mechanical signalsbetween the extracellular matrix and the intracellular actin cytoskeleton, and the tensiondictates the coupling of further adaptor and signaling proteins to talin. These mechanical andbiochemical signals affect the shape and motion of the cell, and regulate gene expression.Despite numerous studies on talin-integrin interactions, it is not yet fully clear how talinactivates integrin, and the complete structure of the integrin-binding talin head domainremains to be solved.In this thesis I focus on the interactions of integrin and the talin head domain using moleculardynamics simulations. In the first study we analyzed how mechanical load affects the talin-integrin binding interface in a set of constant force pulling simulations. We found that themechanical stability of the talin-integrin interface requires a set of anti-parallel β-sheet-likehydrogen bonds, but not the binding of the β-integrin NPxY motif.In the second and third studies, we studied the talin-αIIbβ3 integrin complex in the presenceof a lipid bilayer. These simulations were carried out using two different talin models: anextended talin head conformation based on a published crystallographic structure, and ourproposed compact talin conformation, whose design is based on homologous 3-D structures.Simulations of the extended talin conformation showed that the integrin dimer could becomeperturbed, and even activated, by acidic phospholipids in the cell membrane. With our own,compact domain model, our MD simulations predicted an interaction between a flexible loopand integrin. Experiments carried outin vitro andin cellulo supported the proposed compactconformation of the talin head and provided evidence for the predicted interaction betweenthe talin loop and integrin

Bacterial avidins are a widely distributed protein family in Actinobacteria, Proteobacteria and Bacteroidetes

Background: Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptavidin from Streptomyces avidinii, a growing number of avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for avidin encoding genes among bacteria using available databases and classified potential avidins according to taxonomy and the ecological niches utilized by host bacteria. Results: Numerous avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding avidins were found. The living strategies of bacteria hosting avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. Conclusions: Bacterial avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.publishedVersionPeer reviewe