Browsing by Subject "PROTEINS"

Sort by: Order: Results:

Now showing items 1-20 of 98
  • Jylhava, Juulia; Lyytikainen, Leo-Pekka; Kahonen, Mika; Hutri-Kahonen, Nina; Kettunen, Johannes; Viikari, Jorma; Raitakari, Olli T.; Lehtimaki, Terho; Hurme, Mikko (2012)
  • Salo, Tuula; Sutinen, Meeri; Apu, Ehsanul Hoque; Sundquist, Elias; Cervigne, Nilva K.; de Oliveira, Carine Ervolino; Akram, Saad Ullah; Ohlmeier, Steffen; Suomi, Fumi; Eklund, Lauri; Juusela, Pirjo; Astrom, Pirjo; Bitu, Carolina Cavalcante; Santala, Markku; Savolainen, Kalle; Korvala, Johanna; Paes Leme, Adriana Franco; Coletta, Ricardo D. (2015)
    Background: The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel (R), are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel (R) is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. Methods: A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel (R). Myogel was tested and compared to Matrigel (R) in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. Results: We demonstrated that only 34 % of Myogel's molecular content was similar to Matrigel (R). All test results showed that Myogel was comparable with Matrigel (R), and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel (R) in in vitro Transwell (R) invasion and capillary formation assays. Conclusions: In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.
  • Konttinen, Y T; Mandelin, J; Li, T F; Salo, J; Lassus, J; Liljestrom, M; Hukkanen, M; Takagi, M; Virtanen, I; Santavirta, S (2002)
  • Kontturi, Miia; Junni, Reijo; Kujala-Wirth, Minna; Malinen, Erja; Seuna, Eija; Pelkonen, Sinikka; Soveri, Timo; Simojoki, Heli (2020)
    Several Finnish dairy herds have suffered from outbreaks of interdigital phlegmon (IP). In these new types of outbreaks, morbidity was high and clinical signs severe, resulting in substantial economic losses for affected farms. In our study, we visited 18 free stall dairy herds experiencing an outbreak of IP and 3 control herds without a similar outbreak. From a total of 203 sampled cows, 60 suffered from acute stage IP. We demonstrated that acute phase response of bovine IP was evident and therefore an appropriate analgesic should be administered in the treatment of affected animals. The response was most apparent in herds with high morbidity in IP and with a bacterial infection comprising Fusobacterium necrophorum and Dichelobacter nodosus, indicating that combination of these two bacterial species affect the severity of the disease.
  • Zhang, Yuezhou; Jumppanen, Antti Mikael; Maksimainen, Mirko M.; Auno, Atte Samuli; Awol, Zulfa; Ghemtio, Leo; Venkannagari, Harikanth; Lehtiö, Lari; Yli-Kauhaluoma, Jari; Xhaard, Henri; Boije af Gennäs, Gustav (2018)
    The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 50-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations. (C) 2018 Elsevier Ltd. All rights reserved.
  • Romano, Roberta; Rivellini, Cristina; De Luca, Maria; Tonlorenzi, Rossana; Beli, Raffaella; Manganelli, Fiore; Nolano, Maria; Santoro, Lucio; Eskelinen, Eeva-Liisa; Previtali, Stefano C.; Bucci, Cecilia (2021)
    The small GTPase RAB7A regulates late stages of the endocytic pathway and plays specific roles in neurons, controlling neurotrophins trafficking and signaling, neurite outgrowth and neuronal migration. Mutations in the RAB7A gene cause the autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) disease, an axonal peripheral neuropathy. As several neurodegenerative diseases are caused by alterations of endocytosis, we investigated whether CMT2B-causing mutations correlate with changes in this process. To this purpose, we studied the endocytic pathway in skin fibroblasts from healthy and CMT2B individuals. We found higher expression of late endocytic proteins in CMT2B cells compared to control cells, as well as higher activity of cathepsins and higher receptor degradation activity. Consistently, we observed an increased number of lysosomes, accompanied by higher lysosomal degradative activity in CMT2B cells. Furthermore, we found increased migration and increased RAC1 and MMP-2 activation in CMT2B compared to control cells. To validate these data, we obtained sensory neurons from patient and control iPS cells, to confirm increased lysosomal protein expression and lysosomal activity in CMT2B-derived neurons. Altogether, these results demonstrate that in CMT2B patient-derived cells, the endocytic degradative pathway is altered, suggesting that higher lysosomal activity contributes to neurodegeneration occurring in CMT2B.
  • Colecchia, D; Stasi, M; Leonardi, M; Manganelli, F; Nolano, M; Veneziani, BM; Santoro, L; Eskelinen, Eeva-Liisa; Chiariello, M; Bucci, Cecilia (2018)
    Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.
  • Hanski, Leena; Ausbacher, Dominik; Tiirola, Terttu M.; Strom, Morten B.; Vuorela, Pia M. (2016)
    We demonstrate in the current work that small cationic antimicrobial beta(2,2)-amino acid derivatives (Mw <500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (<5 mu M, i.e. <3.4 mu g/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the beta(2,2)-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these beta(2,2)-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic beta(2,2)-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-totreat intracellular pathogen.
  • Li, Shiqian; Prasanna, Xavier; Salo, Veijo T.; Vattulainen, Ilpo; Ikonen, Elina (2019)
    Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron minilAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.
  • Wang, Xin; Ye, Lingling; Lyu, Munan; Ursache, Robertas; Löytynoja, Ari; Mähönen, Ari Pekka (2020)
    Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type-specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a new tool with which target genes can efficiently and conditionally be knocked out by genome editing at any developmental stage. Target genes can also be knocked out in a cell-type-specific manner. Our tool is easy to construct and will be particularly useful for studying genes having null alleles that are non-viable or show pleiotropic developmental defects.
  • Rooijers, Koos; Kolmeder, Carolin; Juste, Catherine; Dore, Joel; de Been, Mark; Boeren, Sjef; Galan, Pilar; Beauvallet, Christian; de Vos, Willem M.; Schaap, Peter J. (2011)
  • Levanova, Alesia; Poranen, Minna Marjetta (2018)
    Steric exclusion chromatography (SXC) is a method for separation of large target solutes based on their association with a hydrophilic stationary phase through mutual steric exclusion of polyethylene glycol (PEG). Selectivity in SXC is determined by the size or shape (or both) of the solutes alongside the size and concentration of PEG molecules. Elution is achieved by decreasing the PEG concentration. In this study, SXC applicability for the separation and purification of single-stranded (ss) and double-stranded (ds) RNA molecules was evaluated for the first time. The retention of ssRNA and dsRNA molecules of different lengths on convective interaction media (CIM) monolithic columns was systematically studied under variable PEG-6000 and NaCl concentrations. We determined that over 90% of long ssRNAs (700-6374 nucleotides) and long dsRNAs (500-6374 base pairs) are retained on the stationary phase in 15% PEG-6000 and >= 0.4 M NaCl. dsDNA and dsRNA molecules of the same length were partially separated by SXC. Separation of RNA molecules below 100 nucleotides from longer RNA species is easily achieved by SXC. Furthermore, SXC has the potential to separate dsRNAs from ssRNAs of the same length. We also demonstrated that SXC is suitable for the enrichment of ssRNA (PRR1 bacteriophage) and dsRNA (Phi6 bacteriophage) viral genomes from contaminating cellular RNA species. In summary, SXC on CIM monolithic columns is an appropriate tool for rapid RNA separation and concentration. (C) 2018 The Authors. Published by Elsevier B.V.
  • Myllymaki, Satu-Marja; Kämäräinen, Ulla-Reetta; Liu, Xiaonan; Cruz, Sara Pereira; Miettinen, Sini; Vuorela, Mikko; Varjosalo, Markku; Manninen, Aki (2019)
    Integrin-mediated laminin adhesions mediate epithelial cell anchorage to basement membranes and are critical regulators of epithelial cell polarity. Integrins assemble large multiprotein complexes that link to the cytoskeleton and convey signals into the cells. Comprehensive proteomic analyses of actin network-linked focal adhesions (FA) have been performed, but the molecular composition of intermediate filament-linked hemidesmosomes (HD) remains incompletely characterized. Here we have used proximity-dependent biotin identification (BioID) technology to label and characterize the interactome of epithelia-specific beta 4-integrin that, as alpha 6 beta 4-heterodimer, forms the core of HDs. The analysis identified similar to 150 proteins that were specifically labeled by BirA-tagged integrin-beta 4. In addition to known HDs proteins, the interactome revealed proteins that may indirectly link integrin-beta 4 to actin-connected protein complexes, such as FAs and dystrophin/dystroglycan complexes. The specificity of the screening approach was validated by confirming the HD localization of two candidate beta 4-interacting proteins, utrophin (UTRN) and ELKS/Rab6-interacting/CAST family member 1 (ERC1). Interestingly, although establishment of functional HDs depends on the formation of alpha 6 beta 4-heterodimers, the assembly of beta 4-interactome was not strictly dependent on alpha 6-integrin expression. Our survey to the HD interactome sets a precedent for future studies and provides novel insight into the mechanisms of HD assembly and function of the beta 4-integrin.
  • Rebbeck, Timothy R.; Mitra, Nandita; Wan, Fei; Sinilnikova, Olga M.; Healey, Sue; McGuffog, Lesley; Mazoyer, Sylvie; Chenevix-Trench, Georgia; Easton, Douglas F.; Antoniou, Antonis C.; Nathanson, Katherine L.; CIMBA Consortium; Nevanlinna, Heli; Aittomäki, Kristiina (2015)
    IMPORTANCE Limited information about the relationship between specific mutations in BRCA1 or BRCA2 (BRCA1/2) and cancer risk exists. OBJECTIVE To identify mutation-specific cancer risks for carriers of BRCA1/2. DESIGN, SETTING, AND PARTICIPANTS Observational study of women who were ascertained between 1937 and 2011 (median, 1999) and found to carry disease-associated BRCA1 or BRCA2 mutations. The international sample comprised 19 581 carriers of BRCA1 mutations and 11 900 carriers of BRCA2 mutations from 55 centers in 33 countries on 6 continents. We estimated hazard ratios for breast and ovarian cancer based on mutation type, function, and nucleotide position. We also estimated RHR, the ratio of breast vs ovarian cancer hazard ratios. A value of RHR greater than 1 indicated elevated breast cancer risk; a value of RHR less than 1 indicated elevated ovarian cancer risk. EXPOSURES Mutations of BRCA1 or BRCA2. MAIN OUTCOMES AND MEASURES Breast and ovarian cancer risks. RESULTS Among BRCA1 mutation carriers, 9052 women (46%) were diagnosed with breast cancer, 2317(12%) with ovarian cancer, 1041 (5%) with breast and ovarian cancer, and 7171 (37%) without cancer. Among BRCA2 mutation carriers, 6180 women (52%) were diagnosed with breast cancer, 682(6%) with ovarian cancer, 272(2%) with breast and ovarian cancer, and 4766 (40%) without cancer. In BRCA1, we identified 3 breast cancer cluster regions (BCCRs) located at c.179 to c.505 (BCCR1; RHR = 1.46; 95% Cl, 1.22-1.74; P = 2 x 10(-6)), c.4328 to c.4945 (BCCR2; RH R = 1.34; 95% Cl, 1.01-1.78; P =.04), and c. 5261 to c.5563 (BCCR2', RHR = 1.38; 95% Cl, 1.22-1.55; P = 6 x 10(-9)). We also identified an ovarian cancer cluster region (OCCR) from c.1380 to c.4062 (approximately exon 11) with RHR = 0.62 (95% Cl, 0.56-0.70; P = 9 x 10(-17)). In BRCA2, we observed multiple BCCRs spanning c.1 to c.596 (BCCR1; RHR = 1.71; 95% Cl, 1.06-2.78; P =.03), c.772 to c.1806 (BCCRI; RHR = 1.63; 95% Cl, 1.10-2.40; P =.01), and c.7394 to c.8904 (BCCR2; RHR = 2.31; 95% Cl, 1.69-3.16; P =.00002). We also identified 3 OCCRs: the first (OCCR1) spanned c.3249 to c.5681 that was adjacent to c.5946delT (6174delT; RHR = 0.51; 95% Cl, 0.44-0.60; P = 6 x 10(-17)). The second OCCR spanned c.6645 to c.7471 (OCCR2; RHR = 0.57; 95% Cl, 0.41-0.80; P =.001). Mutations conferring nonsense-mediated decay were associated with differential breast or ovarian cancer risks and an earlier age of breast cancer diagnosis for both BRCA1 and BRCA2 mutation carriers. CONCLUSIONS AND RELEVANCE Breast and ovarian cancer risks varied by type and location of BRCA1/2 mutations. With appropriate validation, these data may have implications for risk assessment and cancer prevention decision making for carriers of BRCA1 and BRCA2 mutations.
  • Toledo, Miriam; Batista-Gonzalez, Ana; Merheb, Emilio; Aoun, Marie Louise; Tarabra, Elena; Feng, Daorong; Sarparanta, Jaakko; Merlo, Paola; Botre, Francesco; Schwartz, Gary J.; Pessin, Jeffrey E.; Singh, Rajat (2018)
    The circadian clock coordinates behavioral and circadian cues with availability and utilization of nutrients. Proteasomal degradation of clock repressors, such as cryptochrome (CRY) 1, maintains periodicity. Whether macroautophagy, a quality control pathway, degrades circadian proteins remains unknown. Here we show that circadian proteins BMAL1, CLOCK, REV-ERB alpha, and CRY1 are lysosomal targets, and that macroautophagy affects the circadian clock by selectively degrading CRY1. Autophagic degradation of CRY1, an inhibitor of gluconeogenesis, occurs in a diurnal window when rodents rely on gluconeogenesis, suggesting that CRY1 degradation is timeimprinted to maintenance of blood glucose. High-fat feeding accelerates autophagic CRY1 degradation and contributes to obesity-associated hyperglycemia. CRY1 contains several light chain 3 (LC3)-interacting region (LIR) motifs, which facilitate the interaction of cargo proteins with the autophagosome marker LC3. Using mutational analyses, we identified two distinct LIRs on CRY1 that exert circadian glycemic control by regulating CRY1 degradation, revealing LIRs as potential targets for controlling hyperglycemia.
  • Pahr, Sandra; Selb, Regina; Weber, Milena; Focke-Tejkl, Margarete; Hofer, Gerhard; Dordic, Andela; Keller, Walter; Papadopoulos, Nikolaos G.; Giavi, Stavroula; Makela, Mika; Pelkonen, Anna; Niederberger, Verena; Vrtala, Susanne; Valenta, Rudolf (2014)
  • Ligthart, Kate; Belzer, Clara; de Vos, Willem M.; Tytgat, Hanne L.P. (2020)
    Cell-surface-located proteinaceous appendages, such as flagella and fimbriae or pili, are ubiquitous in bacterial communities. Here, we focus on conserved type IV pili (T4P) produced by bacteria in the intestinal tract, one of the most densely populated human ecosystems. Computational analysis revealed that approximately 30% of known intestinal bacteria are predicted to produce T4P. To rationalize how T4P allow intestinal bacteria to interact with their environment, other microbiota members, and host cells, we review their established role in gut commensals and pathogens with respect to adherence, motility, and biofilm formation, as well as protein secretion and DNA uptake. This work indicates that T4P are widely spread among the known members of the intestinal microbiota and that their contribution to human health might be underestimated.
  • Ciuba, Katarzyna; Hawkes, William; Tojkander, Sari; Kogan, Konstantin; Engel, Ulrike; Iskratsch, Thomas; Lappalainen, Pekka (2018)
    Contractile actomyosin bundles, stress fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), stress fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with stress fibers. However, whether Cnn3 promotes stress fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral stress fibers and transverse arcs, as well as to non-contractile dorsal stress fibers that do not contain NMII. Fluorescencerecovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of stress fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for stress fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of stress fibers that often led to breakage of individual actomyosin bundles within the stress fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the stress fiber network.
  • Najumudeen, A. K.; Jaiswal, A.; Lectez, B.; Oetken-Lindholm, C.; Guzman, C.; Siljamaki, E.; Posada, I. M. D.; Lacey, E.; Aittokallio, T.; Abankwa, D. (2016)
    Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.