A CELL-FREG SYSTEM OF PROTEIN SYNTHESIS FROM MOUSE (C3HB) LIVER

A CELL FREE SYSTEM OF PROTEIN SYNTHESIS OF MOUSE LIVER WAS DEVELOPED. A MOST INTERESTING FEATURE OF THIS SYSTEM IS ITS VERY HIGH ACTIVITY WITH ENDOGENOUS MRNA. A WIDE RANGE OF PRODUCTS ARE LABELLED DURING OPERATION OF THE SYSTEM. THERE IS A LAG PERIOD IN THE LABELLING OF SMALL AND HIGH MOLECULAR WEIGHT PRODUCTS. CYCLOBEXIMIDE, WHICH INHIBITS OVERALL PROTEIN SYNTHESIS BY 80% ALMOST COMPLETELY INHIBITS SYNTHESIS OF PROTEINS APPEARING LATE. OF THE POLYAMINES TESTED SPERMIDINE WEAKLY ACTIVATES AT LOW CONCENTRATIONS AND SPERMINE IS HIGHLY INHIBITORY EVEN AT LOW CONCENTRATIONS. INCREASING CONCENTRATIONS OF SPERMIDINE INTENSIFY LABELLING OF AT LEAST TWO PROTEINS WITH M.W 14.5 AND 15.5 KD. EXOGENOUS MRNA (GLOBIN MRNA, TMV RNA) CAN BE TRANSLATED IF THE SYSTEM IS TREATED EITHER BY MICROCOCCAL NUCLEASE OR BY PREINCUBATION TO EXHAUST ENDOGENOUS MRNA. IN THESECOND CASE THE PREINCUBATED SYSTEM HAS TO BE SUPPLIED WITH ADDITIONAL INITIATION FACTORS. AN INHIBITORY AGENT IS EXTRACTED WITH THE INITIATION FACTORS. THE INHIBITOR WAS PURIFIED WITH DEAE-BIOGEL A, SEPHADEX C50 CONCLUDED THAT THE RESPONSIBLE AGENT IS MOST LIKELY TRACES OF AMINOACIDS THAT DILUTE THE LABELLEDAMINOACID.ΑΝΑΠΤΥΧΘΗΚΕ ΕΝΑ ΣΥΣΤΗΜΑ ΠΡΩΤΕΙΝΟΣΥΝΘΕΣΗΣ ΕΛΕΥΘΕΡΟ ΚΥΤΤΑΡΩΝ ΑΠΟ ΣΥΚΩΤΙ ΠΟΝΤΙΚΩΝC3HB, ΜΕ ΚΥΡΙΟ ΧΑΡΑΚΤΗΡΙΣΤΙΚΟ ΤΗΝ ΥΨΗΛΗ ΕΝΔΟΓΕΝΗ ΔΡΑΣΗ. ΑΠΟ ΑΥΤΟΡΑΔΙΟΓΡΑΦΙΑ ΠΡΟΚΥΠΤΕΙ ΟΤΙ, ΜΕ ΤΟ ΕΝΔΟΓΕΝΕΣ MRNA, ΣΥΝΘΕΤΕΤΑΙ ΕΝΑΣ ΜΕΓΑΛΟΣ ΑΡΙΘΜΟΣ ΠΡΟΙΟΝΤΩΝ ΜΕ Μ.Β. ΜΕΓΑΛΥΤΕΡΑ ΤΩΝ 100.000 ΕΩΣ 10.000. ΟΡΙΣΜΕΝΑ ΑΠΟ ΑΥΤΑ ΕΜΦΑΝΙΖΟΝΑΙ ΠΡΩΙΜΑ ΣΤΗ ΚΑΜΠΥΛΗ ΧΡΟΝΟΥ ΕΝΩ ΑΛΛΩΝ Η ΕΜΦΑΝΙΣΗ ΚΑΘΥΣΤΕΡΕΙ. ΠΡΟΣΘΗΚΗ ΚΥΚΛΟΕΞΙΜΙΔΙΟΥ ΔΙΝΕΙ 80% ΑΝΑΣΤΟΛΗ. ΟΙ ΠΡΩΤΕΙΝΕΣ ΠΟΥ ΑΝΑΣΤΕΛΛΟΝΤΑΙ ΚΥΡΙΩΣ ΕΙΝΑΙ ΕΚΕΙΝΕΣ ΠΟΥ ΚΑΘΥΣΤΕΡΟΥΝ ΣΤΗΝ ΚΑΜΠΥΛΗ ΧΡΟΝΟΥ. ΠΡΟΣΘΗΚΗ ΣΠΕΡΜΙΔΙΝΗΣ ΣΕ ΣΥΓΚΕΝΤΡΩΣΕΙΣ 100-500 ΜΜ ΠΡΟΚΑΛΕΙ ΕΝΕΡΓΟΠΟΙΗΣΗ ΕΝΩ ΣΕ ΜΕΓΑΛΥΤΕΡΕΣ ΣΥΓΚΕΝΤΡΩΣΕΙΣ ΠΡΟΚΑΛΕΙ ΑΝΑΣΤΟΛΗ. ΗΣΠΕΡΜΙΝΗ ΠΡΟΚΑΛΕΙ ΜΕΓΑΛΗ ΑΝΑΣΤΟΛΗ. ΠΑΡΟΥΣΙΑ ΤΩΝ ΑΝΑΣΤΑΛΤΙΚΩΝ ΣΥΓΚΕΝΤΡΩΣΕΩΝ ΤΗΣ ΣΠΕΡΜΙΔΙΝΗΣ ΔΥΟ ΠΡΩΤΕΙΝΕΣ ΜΕ Μ.Β 14,5 ΚΑΙ 15,5 KD ΦΑΙΝΕΤΑΙ ΝΑ ΙΣΧΥΡΟΠΟΙΟΥΝΤΑΙ. ΤΟ ΣΥΣΤΗΜΑ ΕΧΕΙ ΤΗΝ ΙΚΑΝΟΤΗΤΑ ΝΑ ΜΕΤΑΦΡΑΖΕΙ ΕΞΩΓΕΝΗ MRNA (MRNA ΣΦΑΙΡΙΝΗΣ, TMV RNA) ΕΙΤΕ ΜΕΤΑ ΑΠΟ ΚΑΤΕΡΓΑΣΙΑ ΜΕ ΜΙΚΡΟΚΟΚΚΙΚΗ ΝΟΥΚΛΕΑΣΗ ΕΙΤΕ ΜΕΤΑ ΑΠΟ ΠΡΟΕΠΩΑΣΗ ΓΙΑ ΤΗΝ ΕΞΑΝΤΛΗΣΗ ΤΟΥ ΕΝΔΟΓΕΝΟΥΣ MRNA. ΣΤΗ ΔΕΥΤΕΡΗ ΠΕΡΙΠΤΩΣΗ ΑΠΑΡΑΙΤΗΤΗ ΕΙΝΑΙ Η ΠΡΟΣΘΗΚΗ ΠΑΡΑΓΟΝΤΩΝ ΕΝΑΡΞΗΣ. ΜΑΖΙ ΜΕ ΤΟΥΣ ΠΑΡΑΓΟΝΤΕΣ ΕΝΑΡΞΗΣ ΕΚΧΥΛΙΖΕΤΑΙ ΚΑΙ ΕΝΑΣ ΑΝΑΣΤΟΛΕΑΣ, ΠΟΥ ΜΕΤΑ ΑΠΟ ΚΑΘΑΡΙΣΜΟ ΣΕ DEAE-BIOGEL A, SEPHADEX G-50, ΚΑΙ BIOGEL P2 ΔΙΑΠΙΣΤΩΘΗΚΕ ΟΤΙ ΠΡΟΚΕΙΤΑΙ ΓΙΑ ΜΙΚΡΑ ΠΟΣΑ ΑΜΙΝΟΞΕΩΝ ΠΟΥ ΑΠΛΩΣ ΑΡΑΙΩΝΟΥΝ ΤΟ ΑΝΤΙΣΤΟΙΧΟ ΡΑΔΙΕΝΕΡΓΟ ΑΜΙΝΟΞΥ

Lamin B Receptor: Interplay between Structure, Function and Localization

Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophobic segments that span the membrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have been attributed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contributing to the shape of interphase nuclear architecture, while its transmembrane domains exhibit sterol reductase activity. Mutations within the transmembrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huët anomaly and Greenberg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic behavior have not yet been fully unraveled. Here, we provide an overview of the current knowledge of the interplay between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR

SR Protein Kinase 1 Inhibition by TAF15

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins

SR Protein Kinase 1 Inhibition by TAF15

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases’ partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins

Temporal Association of Protamine 1 with the Inner Nuclear Membrane Protein Lamin B Receptor during Spermiogenesis

International audienceDuring mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32

The O-β-linked N-acetylglucosaminylation of the Lamin B receptor and its impact on DNA binding and phosphorylation

International audienc

Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates

Peptide–drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανβ3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases