Commander Complex—A Multifaceted Operator in Intracellular Signaling and Cargo

Commander complex is a 16-protein complex that plays multiple roles in various intracellular events in endosomal cargo and in the regulation of cell homeostasis, cell cycle and immune response. It consists of COMMD1–10, CCDC22, CCDC93, DENND10, VPS26C, VPS29, and VPS35L. These proteins are expressed ubiquitously in the human body, and they have been linked to diseases including Wilson’s disease, atherosclerosis, and several types of cancer. In this review we describe the function of the commander complex in endosomal cargo and summarize the individual known roles of COMMD proteins in cell signaling and cancer. It becomes evident that commander complex might be a much more important player in intracellular regulation than we currently understand, and more systematic research on the role of commander complex is required

Commander Complex—A Multifaceted Operator in Intracellular Signaling and Cargo

Commander complex is a 16-protein complex that plays multiple roles in various intracellular events in endosomal cargo and in the regulation of cell homeostasis, cell cycle and immune response. It consists of COMMD1–10, CCDC22, CCDC93, DENND10, VPS26C, VPS29, and VPS35L. These proteins are expressed ubiquitously in the human body, and they have been linked to diseases including Wilson’s disease, atherosclerosis, and several types of cancer. In this review we describe the function of the commander complex in endosomal cargo and summarize the individual known roles of COMMD proteins in cell signaling and cancer. It becomes evident that commander complex might be a much more important player in intracellular regulation than we currently understand, and more systematic research on the role of commander complex is required

The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.Hedgehog (Hh) -perheen kasvutekijät säätelevät alkion kehitystä ja kudosten erilaistumista niin selkärankaisissa kuin selkärangattomissakin eläinlajeissa. Hhkasvutekijää koodaava geeni löydettiin vuonna 1992 banaanikärpäsestä (Drosophila melanogaster), jonka jälkeen tämän geeniperheen geenejä on löydetty useista muistakin eliölajeista –hyönteisistä nisäkkäisiin. Ihmisellä Hh geenejä on kolme: Sonic (Shh), Indian (Ihh) ja Desert (Dhh). Hedgehog kasvutekijän signaalinvälitykselle ovat tyypillisiä useat molekyylien väliset estävät vuorovaikutukset. Hh kasvutekijän sitoutuminen reseptoriproteiini Patchediin (Ptc) estää Ptc:n toiminnan ja vapauttaa signaalinvälittäjäproteiini Smoothenedin (Smo) Ptc:n estävästä vaikutuksesta. Useat Hh signaalinvälitykseen osallistuvat molekyylit on alunperin löydetty banaanikärpäsestä, ja nisäkkäiden Hh signaalinvälitykseen osallistuvista sytoplasmisista komponenteista on tunnistettu vain transkriptiotekijä Cubitus interruptusta (Ci) vastaavat GLI(1-3), ja Ci/GLI proteiineja sitova Suppressor of Fused (Su(Fu)). Tämän tutkimuksen tarkoituksena oli selvittää kuinka Shh-signaali välittyy solun pinnalta solun sisään johtaen lopulta GLI transkriptiotekijöiden aktivoitumiseen ja signaalinvälitysreitin kohdegeenien ilmentymiseen. Banaanikärpäsessä kinesiinin-kaltainen proteiini Costal2 (Cos2) säätelee keskeisesti signaalinvälitysreitin toimintaa ja Cos2 geenin puute johtaa kärpäsessä toukan kehityksen häiriintymiseen ja kuolemaan. Myös toinen proteiini, Su(Fu) osallistuu Ci-transkriptiotekijän aktiivisuuden hienosäätöön banaanikärpäsessä, mutta sen puutos aiheuttaa vain hyvin pieniä muutoksia siiven rakenteessa. Tutkimuksessamme havaitsimme yllättäen, että Hh-signaalinvälitysreitin toiminta eroaa merkittävästi nisäkkäiden ja kärpäsen välillä, alkaen heti solun pinnalla olevan Smo proteiinin rakenteesta. Eristettyämme Cos2 geenin nisäkäshomologit huomasimme myös, ettei niiden toimintaa tarvita nisäkkäissä. Nisäkkäissä signaalireitin aktiivisuutta sääteleekin keskeisesti Su(Fu). Tuloksemme osoittavat, että muuttumattomina pidetyt signaalinvälitysreitit voivat muuttua evoluution aikana. Löytääksemme uusia nisäkkäiden Hh-signaalinvälitysreitin toimintaa sääteleviä geenejä, eristimme myös yli 90% kaikista ihmisen proteiinikinaaseista ja analysoimme niiden merkitystä Shhsignaalireitin toiminnalle tehoseulontamenetelmin. Näin pystyimme löytämään kaksi uutta Shh-signaalireitin toimintaa säätelevää proteiinikinaasia, MAP3K10 ja DYRK2. Havaitsimme, että DYRK2 vaikuttaa suoraan GLI-transkriptiotekijöihin fosforyloimalla niitä ja johtaen näin niiden hajotukseen. MAP3K10 puolestaan vaikuttaa GLItranskriptiotekijöihin epäsuorasti, säätelemällä muiden kinaasien, mm. DYRK2:n, toimintaa

Human transcription factor and protein kinase gene fusions in human cancer

Oncogenic gene fusions are estimated to account for up-to 20% of cancer morbidity. Recently sequence-level studies have established oncofusions throughout all tissue types. However, the functional implications of the identified oncofusions have often not been investigated. In this study, identified oncofusions from a fusion detection approach (DEEPEST) were analyzed in detail. Of the 28,863 oncofusions, we found almost 30% are expected to produce functional proteins with features from both parent genes. Kinases and transcription factors were the main gene families of the protein producing fusions. Considering their role as initiators, actors, and termination points of cellular signaling pathways, we focused our in-depth analyses on them. Domain architecture of the fusions and their wild-type interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins. To understand overall oncofusion effects, we performed differential expression analysis using TCGA cancer project samples. Results indicated oncofusion-specific alterations in gene expression levels, and lower expression levels of components of key cellular pathways, in particular signal transduction and transcription regulation. The sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling, possibly via acquiring novel functions.Peer reviewe

Ribosome profiles and riboproteomes of healthy and Potato virus A- and Agrobacterium-infected Nicotiana benthamiana plants

Nicotiana benthamiana is an important model plant for plant-microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants.Peer reviewe

Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

In this study, we investigated the significance of a conserved five-amino acid motif 'AELPR' in the C-terminal region of helper component-proteinase (HCPro) for potato virus A (PVA; genusPotyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCPro(WD). These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCPro(WD)to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCPro(WD)(PVA(WD)) infectedNicotiana benthamianaless efficiently than wild-type PVA (PVA(WT)) and HCPro(WD)complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCPro(WD). Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCPro(WD)-containing HMW-complexes. A boost in translation of replication-deficient PVA (PVA(Delta GDD)) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derivedRenillaluciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVA(WD)was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multi-protein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection. Author summary This study revealed that the potyviral helper component proteinase (HCPro) and the host protein VARICOSE (VCS) are linked in a manner that is important for suppression of RNA silencing, formation of potyvirus-induced RNA granules, translation of viral proteins, stability of virions, and development of systemic potato virus A (PVA) infection. The results suggest that HCPro and VCS belong to the core components of large RNP complexes regulating PVA infection. We suggest that these complexes protect viral RNAs in the cytoplasm after release from the replication complex and direct them to translation and intact to the viral particles.Peer reviewe

Talin-mediated force transmission and talin rod domain unfolding independently regulate adhesion signaling

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between beta-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.Peer reviewe

Skeletal muscle proteomes reveal downregulation of mitochondrial proteins in transition from prediabetes into type 2 diabetes

Skeletal muscle insulin resistance is a central defect in the pathogenesis of type 2 diabetes (T2D). Here, we analyzed skeletal muscle proteome in 148 vastus lateralis muscle biopsies obtained from men covering all glucose tolerance phenotypes: normal, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and T2D. Skeletal muscle proteome was analyzed by a sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics technique. Our data indicate a downregulation in several proteins involved inmitochondrial electron transport or respiratory chain complex assembly already in IFG and IGT-muscles, with most profound decreases observed in T2D. Additional phosphoproteomic analysis reveals altered phosphorylation in several signaling pathways in IFG, IGT, and T2D muscles, including those regulating glucose metabolic processes, and the structure of muscle cells. These data reveal several alterations present in skeletalmuscle already in prediabetes and highlight impairedmitochondrial energy metabolism in the trajectory from prediabetes into T2D.Peer reviewe

Nuclear proteome of virus-infected and healthy potato leaves

Abstract Background Infection of plants by viruses interferes with expression and subcellular localization of plant proteins. Potyviruses comprise the largest and most economically damaging group of plant-infecting RNA viruses. In virus-infected cells, at least two potyviral proteins localize to nucleus but reasons remain partly unknown. Results In this study, we examined changes in the nuclear proteome of leaf cells from a diploid potato line (Solanum tuberosum L.) after infection with potato virus A (PVA; genus Potyvirus; Potyviridae) and compared the data with that acquired for healthy leaves. Gel-free liquid chromatography–coupled to tandem mass spectrometry was used to identify 807 nuclear proteins in the potato line v2–108; of these proteins, 370 were detected in at least two samples of healthy leaves. A total of 313 proteins were common in at least two samples of healthy and PVA-infected leaves; of these proteins, 8 showed differential accumulation. Sixteen proteins were detected exclusively in the samples from PVA-infected leaves, whereas other 16 proteins were unique to healthy leaves. The protein Dnajc14 was only detected in healthy leaves, whereas different ribosomal proteins, ribosome-biogenesis proteins, and RNA splicing–related proteins were over-represented in the nuclei of PVA-infected leaves. Two virus-encoded proteins were identified in the samples of PVA-infected leaves. Conclusions Our results show that PVA infection alters especially ribosomes and splicing-related proteins in the nucleus of potato leaves. The data increase our understanding of potyvirus infection and the role of nucleus in infection. To our knowledge, this is the first study of the nuclear proteome of potato leaves and one of the few studies of changes occurring in nuclear proteomes in response to plant virus infection