96 research outputs found
MARP protein family: A possible role in molecular mechanisms of tumorigenesis
Familiju MARP (muscle ankyrin repeat proteins) čine tri strukturno slična proteina: CARP/Ankrd1, Ankrd2/Arpp i DARP/Ankrd23. Sva tri proteina poseduju ankirinske ponovke preko kojih ostvaruju protein-protein interakcije kao i signal za lokalizaciju u jedru. Članovi familije MARP imaju strukturnu i regulatornu funkciju i mogu biti lokalizovani i u jedru i u citoplazmi mišićne ćelije. Učestvuju u signalnoj transdukciji kao molekulski glasnici koji prenose informacije mehaničkog stresa sa sarkomere do jedra, gde učestvuju u regulaciji genske ekspresije. Nivo proteina CARP/Ankrd1 i Ankrd2/Arpp je izmenjen u mišićnim bolestima koje karakteriše atrofija mišića, kao što su Dišenova mišićna distrofija, kongenitalna miopatija i spinalna mišićna atrofija. Mutacije u genu za CARP/Ankrd1 su otkrivene u pacijenata sa dilatiranom i hipertrofičnom kardiomiopatijom. Promene u ekspresiji ovih proteina su takođe uočene u tumorima kao što su rabdomiosarkom, onkocitom bubrega i kancer ovarijuma. U cilju funkcionalne karakterizacije proteina familije MARP, pokazali smo da oba proteina interaguju sa supresorom tumora p53, a geni za CARP/Ankrd1 i Ankrd2/Arpp su pozitivno regulisani ovim transkripcionim faktorom. Rezultati su ukazali na moguću ulogu proteina CARP/Ankrd1 i Ankrd2/Arpp u molekularnim mehanizmima tumorogeneze, čime se otvara novo polje istraživanja ove familije proteina.The MARP (muscle ankyrin repeat protein) family comprises three structurally similar proteins: CARP/Ankrd1, Ankrd2/Arpp and DARP/Ankrd23. They share four conserved copies of 33-residue ankyrin repeats and contain a nuclear localization signal, allowing the sorting of MARPs to the nucleus. They are found both in the nucleus and in the cytoplasm of skeletal and cardiac muscle cells, suggesting that MARPs shuttle within the cell enabling them to play a role in signal transduction in striated muscle. Expression of MARPs is altered under different pathological conditions. In skeletal muscle, CARP/Ankrd1 and Ankrd2/Arpp are up-regulated in muscle in patients suffering from Duchene muscular dystrophy, congenital myopathy and spinal muscular atrophy. Mutations in Ankrd1 gene (coding CARP/Ankrd1) were identified in dilated and hypertrophic cardiomyopathies. Altered expression of MARPs is also observed in rhabdomyosarcoma, renal oncocytoma and ovarian cancer. In order to functionally characterize MARP family members CARP/Ankrd1 and Ankrd2/Arpp, we have found that both proteins interact with the tumor suppressor p53 both in vivo and in vitro and that p53 up-regulates their expression. Our results implicate the potential role of MARPs in molecular mechanisms relevant to tumor response and progression
Characterization of human skeletal muscle protein Ankrd2
Predmet istraživanja ovog rada je humani mišićni protein Ankrd2 koji se eksprimira u sporim mišićnim vlaknima i potencijalni je transkripcioni faktor čija je količina povećana u toku hipertrofije skeletnih mišića. To je protein od 37 kDa koji poseduje signal za lokalizaciju u jedru, četiri ankirinska ponovka i veoma je sličan jedarnom proteinu humanih endotelnih ćelija, C-193, čija je ekspresija indukovana citokinima. Pacovski i mišji ortologoni humanog proteina C-193, CARP odnosno MARP su pretežno eksprimirani u srcu i verovatno su uključeni u kontrolu hipertrofije srca. Pretpostavlja se da Ankrd2 u skeletnim mišićima ima sličnu funkciju kao CARP/MARP/C-193 u srcu. Za utvrdjivanje intraćelijske lokalizacije i ekspresije proteina Ankrd2 u različitim tkivima napravljena su tri mišja poliklonska antitela (na ceo protein, N-terminalni i C-terminalni region) i monoklonsko antitelo (na C-terminalni region proteina Ankrd2). Western blot analizom proteinskih ekstrakata različitih humanih tkiva pokazano je da se protein Ankrd2 eksprimira uglavnom u skeletnim mišićima i u manjoj meri u srcu i bubregu. U toku diferencijacije humanih (CHQ5B) i mišjih (C2C12) mišićnih ćelija dolazi do povećanja količine proteina Ankrd2. U cilju detaljne analize intraćelijske lokalizacije proteina Ankrd2 uradjeni su eksperimenti indirektne imunofluorescencije koristeći humane mioblaste i miotube. Generalno, u toku diferencijacije povećava se i broj ćelija koje fluoresciraju i intenzitet signala. Takodje, u humanim mioblastima, za razliku od miotuba, detektovana je specifična jedarna fluorescencija. Jedarni signal je u obliku tački i eksperimenti kolokalizacije su pokazali da se proteini Ankrd2 i PML nalaze u istim jedarnim strukturama nazvanim PML jedarna tela. U cilju izučavanja proteinsko-proteinskih interakcija pripremljeni su rekombinantni proteini fuzionisani sa GST markerom (F-Ankrd2 (5-333 ak), N-Ankrd2 (5-120 ak) i C-Ankrd2 (279-333 ak)) koji su korišćeni u GST „pull-down” eksperimentima. Ovom metodom je pokazano da protein Ankrd2 interaguje sa nekoliko, za sada neidentifikovanih, proteina. U aminokiselinskoj sekvenci proteina Ankrd2 nalaze se četiri potencijalna mesta za fosforilaciju kazein kinazom II (CKII). U ovom radu je pokazano da protein Ankrd2 može biti fosforilisan in vitro ovom kinazom, s tim što je samo četvrto mesto (SGRE, 318-321 ak) fosforilisano. Eksperimenima retardacije DNK na gelu utvrdjeno je da proteini Ankrd2 i IkB, inhibitor transkripcionog faktora NFkB, nemaju sličnu funkciju iako imaju strukturne sličnosti (oba proteina poseduju ankirinske ponovke, signal za lokalizaciju u jedru i mesta za fosforilaciju raznim kinazama). Za razliku od IkB, protein Ankrd2 ne sprečava formiranje kompleksa izmedju DNK i NFkB.The object of this study was the new human muscle protein Ankrd2 found preferentially in slow muscle fibers that is possibly a transcription factor up-regulated in hypertrophy. It is a protein of 37 kDa, which has a signal for nuclear targeting, four ankyrin repeat motifs and shows significant similarity to a cytokine inducible nuclear protein C-193 from human endothelial cells. The rat and mouse orthologs of human C-193 called respectively CARP and MARP are mainly expressed in heart and probably involved in the control of cardiac hypertrophy. It is possible that Ankrd2 may play a similar role to CARP/MARP/C-193, but in skeletal muscle rather than heart. For intracellular localization and tissue distribution of Ankrd2 three mouse polyclonal antibodies (raised against the full length, N-terminal and C-terminal regions of the protein) and one monoclonal antibody (raised against C-terminal region of Ankrd2) were made. Western blot analysis of protein extracts isolated from different human tissues showed that Ankrd2 is expressed mainly in human skeletal muscle and to a lesser extent in heart and kidney. During muscle cell differentiation there is an increase of Ankrd2 signal in both mouse (C2C12) and human (CHQ5B) muscle cells that can be detected by Western blot analysis. Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes with the scope of pinpointing the intracellular localization of Ankrd2 protein. In general, during differentiation both the number of fluorescing cells and the intensity of signal increase. Also, in human myoblasts but not in differentiated myotubes very specific nuclear fluorescence can be detected. The nuclear signal is in the form of speckles and co-localization experiments showed that Ankrd2 and PML co-localize in PML nuclear bodies. In order to study protein-protein interactions GST recombinant proteins F-Ankrd2 (5-333 aa), N-Ankrd2 (5-120 aa) and C-Ankrd2 (279-333 aa) were prepared and used for the GST „pull-down” experiments. Results show that Ankrd2 interacts with several proteins, but so far these proteins are not identified. The deduced amino acid sequence of Ankrd2 contains four putative casein kinase II (CKII) phosphorylation sites. Ankrd2 can be phosphorylated in vitro with CKII, but only the fourth site (318-321 aa, SGRE) is phosphorylated. In order to discover wether Ankrd2 has a similar function to IkB, the inhibitor of transcriptional factor NFkB (as they have structural similarities (ankyrin repeats, nuclear localization signal, phosphorylation sites)) electromobility shift assays (EMSA) were performed. Ankrd2 does not prevent the formation of a complex between NFkB and its DNA binding site whereas IkB in the same experiments was able to do so, thus suggesting that Ankrd2 does not function as muscle specific IkB protein
Characterization of antibodies directed against the Ankrd2 human muscle protein
Protein Ankrd2 poseduje ankirinske ponovke, specifično se eksprimira u skeletnim mišićima i srcu i može biti lokalizovan i u jedru i u citoplazmi mišićne ćelije Pošto antitela na ovaj protein nisu komercijalno dostupna, u ovom radu je opisano generisanje i karakterizacija tri mišja poliklonska i jednog monoklonskog antitela dobijenih na ceo, kao i na amino-terminalni i karboksi-terminalni deo proteina. Korišćenjem rekombinantnih deletanata mapiran je epitop monoklonskog antitela veličine 10 aminokiselina (323-333 ak), a takođe je pokazano da poliklonska antitela na amino-terminalni i karboksiterminalni deo proteina specifično prepoznaju Ankrd2, a ne ankirinske ponovke koji su prisutni u velikom brojuproteina. Sva četiri antitela su visoko specifična za endogeni Ankrd2 koji se eksprimira u srcu i skeletnim mišićima.In order to study the function of the Ankrd2 protein, for which commercial antibodies are not available, we report the production and analysis of polyclonal antibodies to full-length Ankrd2 and its C-terminal and N-terminal regions, as well as a monoclonal antibody to the C-terminus of the protein. Epitope mapping making use of recombinant deletion mutants showed that an epitope located in region 323-333 aa of Ankrd2 is detected by the monoclonal antibody. The high specificity of all four anti-Ankrd2 antibodies for recombinant and endogenous Ankrd2 protein is also demonstrated
The expression of Muscle ankyrin repeat proteins in brown adipose tissue
MARP family members CARP, Ankrd2 and DARP are expressed in the striated muscle, while DARP protein is also detected in brown adipose tissue (BAT). Taking into account recent findings concerning the common origin of muscle and brown fat, expression of CARP and Ankrd2 in mouse BAT was investigated. We demonstrated Ankrd2 expression in both inactive and thermogenically active BAT, while CARP expression was not detected. Our findings suggest that the expression of Ankrd2 in BAT could be a part of the 'myogenic transcriptional signature', further supporting the evidence that muscle and brown adipose cells arise from the same myoblastic precursor
AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp paracasei BGNJ1-64, Functions in Collagen Adhesion, and Pathogen Exclusion In Vitro
Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization
Ankrd1-mediated signaling is supported by its interaction with zonula occludens-1
The muscle ankyrin repeat protein Ankrd1 is localized in a mechanosensory complex of the sarcomeric I-band. It is involved in signaling pathways activated in response to mechanical stretch. It also acts as a transcriptional cofactor in the nucleus, playing an important role in cardiogenesis and skeletal muscle differentiation. To investigate its regulatory function in signaling we employed protein array methodology and identified 10 novel Ankrd1 binding partners among PDZ domain proteins known to act as platforms for multiprotein complex assembly. The zonula occludens protein-1 (ZO-1) was chosen for further analysis since its interaction with Ankrd2 had already been demonstrated. Both Ankrd2 and Ankrd1 have similar functions and localize in the same regions. We confirmed the interaction of Ankrd1 with ZO-1 protein and determined their subcellular distribution in HeLa cells, showing their colocalization in the cytoplasm. Our findings corroborate the role of Ankrd1 in intracellular signaling
Механизми регенерације срца – шта можемо научити од зебрице
Nakon infarkta miokarda povređeno srce čoveka ne može da se regeneriše, već
reaguje formiranjem fibrotičnog ožiljka i remodelovanjem miokarda, koji dovode
do slabljenja njegove funkcije. Miokard sisara je dugo vremena smatran
postmitotičkim i terminalno diferenciranim tkivom. Međutim, kardiomiociti
sisara poseduju ograničenu sposobnost proliferacije. Njihova deoba je redak
događaj i odvija se po veoma niskoj stopi, što svakako nije dovoljno da nadoknadi
milione kardiomiocita trajno izgubljenih usled infarkta miokarda. Trenutno se
u svetu razvijaju dve strategije za podsticanje regeneracije povređenog srca
čoveka kako bi se povratile njegova struktura i funkcija. Jedna strategija
podrazumeva naseljavanje oštećenog tkiva zdravim kardiomiocitima poreklom od
indukovanih pluripotentnih ćelija, dok je cilj druge strategije aktivacija
endogenih mehanizama regeneracije. Za razliku od čoveka, neki kičmenjaci imaju
sposobnost regeneracije povređenih organa, uključujući srce. Životinja koja se
najčešće koristi za proučavanje regeneracije srca je riba zebrica (Danio rerio).
Pionirska studija o izuzetnom regenerativnom kapacitetu srca zebrice nakon
amputacije dela komore objavljena je 2002. godine, nakon koje je usledilo mnoštvo
publikacija o ćelijskim i molekularnim mehanizmima koji doprinose
regenerativnom odgovoru. Regeneracija srca zebrice je rezultat strogo regulisane
interakcije većeg broja procesa, uključujući inflamatorni odgovor,
dediferencijaciju i proliferaciju kardiomiocita, neovaskularizaciju i
reorganizaciju ekstraćelijskog matriksa. Regeneracija se može posmatrati kao
uspavan proces u organima koji ne regenerišu i manipulacijom ovog procesa bi
se mogla postići reaktivacija proliferacije u tim organima nakon povrede.
Ispitivanje interakcije između pro-regenerišućih mehanizama i procesa koji
utiču na regenerativni kapacitet treba da dovede do identifikacije faktora
potrebnih za prevazilaženje blokade regeneracije. Na taj način bi se razvile nove
strategije za indukciju proliferacije kardiomiocita i regeneraciju srca čoveka.Након инфаркта миокарда повређено срце човека не може да се регенерише, већ
реагује формирањем фибротичног ожиљка и ремоделовањем миокарда, који доводе
до слабљења његове функције. Миокард сисара је дуго времена сматран
постмитотичким и терминално диференцираним ткивом. Међутим, кардиомиоцити
сисара поседују ограничену способност пролиферације. Њихова деоба је редак
догађај и одвија се по веома ниској стопи, што свакако није довољно да надокнади
милионе кардиомиоцита трајно изгубљених услед инфаркта миокарда. Тренутно се
у свету развијају две стратегије за подстицање регенерације повређеног срца
човека како би се повратиле његова структура и функција. Једна стратегија
подразумева насељавање оштећеног ткива здравим кардиомиоцитима пореклом од
индукованих плурипотентних ћелија, док је циљ друге стратегије активација
ендогених механизама регенерације. За разлику од човека, неки кичмењаци имају
способност регенерације повређених органа, укључујући срце. Животиња која се
најчешће користи за проучавање регенерације срца је риба зебрица (Danio rerio).
Пионирска студија о изузетном регенеративном капацитету срца зебрице након
ампутације дела коморе објављена је 2002. године, након које је уследило мноштво
публикација о ћелијским и молекуларним механизмима који доприносе
регенеративном одговору. Регенерација срца зебрице је резултат строго регулисане
интеракције већег броја процеса, укључујући инфламаторни одговор,
дедиференцијацију и пролиферацију кардиомиоцита, неоваскуларизацију и
реорганизацију екстраћелијског матрикса. Регенерација се може посматрати као
успаван процес у органима који не регенеришу и манипулацијом овог процеса би
се могла постићи реактивација пролиферације у тим органима након повреде.
Испитивање интеракције између про-регенеришућих механизама и процеса који
утичу на регенеративни капацитет треба да доведе до идентификације фактора
потребних за превазилажење блокаде регенерације. На тај начин би се развиле нове
стратегије за индукцију пролиферације кардиомиоцита и регенерацију срца човека.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022
Noncanonical DNA elements in the lamin B2 origin of DNA replication
DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mM Tris base, 89 mM boric acid, and 2 mM MgCl2) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT) 40 do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads
Ankrd2 in Mechanotransduction and Oxidative Stress Response in Skeletal Muscle: New Cues for the Pathogenesis of Muscular Laminopathies
Ankrd2 (ankyrin repeats containing domain 2) or Arpp (ankyrin repeat, PEST sequence, and proline-rich region) is a member of the muscle ankyrin repeat protein family. Ankrd2 is mostly expressed in skeletal muscle, where it plays an intriguing role in the transcriptional response to stress induced by mechanical stimulation as well as by cellular reactive oxygen species. Our studies in myoblasts from Emery-Dreifuss muscular dystrophy 2, a LMNA-linked disease affecting skeletal and cardiac muscles, demonstrated that Ankrd2 is a lamin A-binding protein and that mutated lamins found in Emery-Dreifuss muscular dystrophy change the dynamics of Ankrd2 nuclear import, thus affecting oxidative stress response. In this review, besides describing the latest advances related to Ankrd2 studies, including novel discoveries on Ankrd2 isoform-specific functions, we report the main findings on the relationship of Ankrd2 with A-type lamins and discuss known and potential mechanisms involving defective Ankrd2-lamin A interplay in the pathogenesis of muscular laminopathies
Functional analysis of novel alpha-1 antitrypsin variants G320R and V321F
Alpha-1 antitrypsin (AAT) gene is highly polymorphic, with a large number of rare variants whose phenotypic consequences often remain inconclusive. Studies addressing functional characteristics of AAT variants are of significant biomedical importance since deficiency and dysfunctionality of AAT are associated with liver and lung diseases. We report the results of the functional analysis of two naturally occurring AAT variants, G320R and V321F, previously identified in patients with lung disease. Neither of variants has been fully functionally characterized. In order to perform their functional analysis both variants were expressed in prokaryotic and eukaryotic systems and their intracellular localization, activity, stability, and polymerization were determined. The results of this study demonstrated that variants G320R and V321F have neither impaired activity against porcine pancreatic elastase nor propensity to form polymers. However, both variants had altered electrophoretic mobility and reduced thermostability when compared to M variant of the protein, indicating a slightly impaired secondary or tertiary structure
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