Study of the biological role and the molecular interactions of a new family of protein kinases that phosphorylate repeating serine-arginine dipeptides

Serine/arginine protein kinases (SRPKs) represent a novel class of enzymes that specifically modify SR or RS dipeptide motifs. In the present thesis we studied the biological role and molecular interactions of a new member of the SRPK family, named SRPK1a. SRPK1a is an alternatively spliced form of SRPK1 and contains an insertion of 171 amino acids at its NH2-terminal domain. This additional sequence of SRPK1a contains a relatively high number of proline residues and two LXXLL motifs (148LAPLL152 and 158LGRLL162), which are thought to facilitate the interaction of different proteins with nuclear receptors. Using the yeast two-hybrid assay, we found that the extended NH2-terminal domain of SRPK1a interacts with the p53 tumor suppressor protein and with Scaffold Attachment Factor-B1, a nuclear scaffold-associated protein that interacts with specific DNA sequences, designated as Matrix Attachment Regions (MARs). Confirmation of the first interaction (SRPK1a with p53) was provided by in vitro pull-down assays, while the second interaction (SRPK1a with SAF-B1) was confirmed by in vitro binding assays, as well as by co-immunoprecipitation from 293T cells doubly transfected with SRPK1a and SAF-B1. Since SAF-B1 and p53 do not contain any RS dipeptides and therefore are not phosphorylated by SRPK1a, we searched for a new molecule linking SAF-B1, p53 and SRPK1a. Our research led us to the identification of P2P-R, a member of the proliferation potential proteins (P2P) that was previously shown to bind p53. P2P-R contains an RS domain that is targeted by SR protein kinases and especially by SRPK1a. Using GST-pulldown and co-immunoprecipitation assays we found that P2P-R also interacts, via its RS region, with SAF-B1. Moreover, both SAF-B1 and P2P-R were found to associate with the DNAse resistant nuclear matrix. In a following step, we showed that both SAF-B1 and P2P-R act as co-repressors of estrogen induced transcription and function as part of the estrogen receptor transcription complex together with estrogen receptor α, steroid receptor co-activator 1 (SRC-1) and co-activator associated arginine methyltransferase (CARM1). Using deletion analysis we found that the repressional activity of P2P-R is confined to its C-terminal most segment (amino acids 1314-1516). Furthermore, using differential cell extraction procedures, we showed that addition of the ERα ligand, estradiol or its antagonist tamoxifen, results in the translocation of a significant part of the receptor to the nuclear matrix fraction. This translocaton coincides with a raise in SR protein kinase activity in the same fraction. Most notably, SRPK1a positively influences estrogen induced transcription. Taken together our observations suggest that nuclear matrix entities influence estrogen-dependent transcription and SRPK1a may modulate the interactions among the components of the estrogen receptor regulatory complex and/or the nuclear matrix, in a manner that leads to transcriptional activation. In addition, we performed studies in HepG2 cells, in collaboration with Dr Hadzopoulou’s group, demonstrating that overexpression of SRPK1a activates HNF-4α-mediated transcription. An SR protein kinase activity was shown to associate tightly with HNF-4α. PGC-1 (PPAR γ Coactivator 1) is the major co-activator of hepatocyte nuclear factor-4α and possesses several repeats of RS dipeptides that are phosphorylated mainly by SRPK1a, and to a lesser extent by SRPK1. In line with this observation, SRPK1a was shown to bind PGC-1 with a much higher affinity than SRPK1. The RS domain of PGC-1 was responsible for this interaction, since GST-PGC-1-ΔRS (a mutant form, lacking the entire RS domain) could not be targeted by the kinase. Overexpression of PGC-1 activates HNF-4α-dependent transcription, whereas the transactivation potential of its mutant form (PGC-1-ΔRS), lacking the RS domain, was reduced by 50%. It is not yet clear whether -and to what extent- the activation of HNF-4α-dependent transcription, by SRPK1a, is mediated by phosphorylation of the RS domain of PGC-1, since SRPK1a is able to impact transcription, even when HepG2 cells were transfected with PGC-1-ΔRS. Finally, in contrast with the estrogen receptor, HNF-4α localization was restricted to the nucleoplasmic fraction of HepG2 cells and there is no evidence for an HNF-4α-based transcriptional complex associated with the nuclear matrix. In agreement with those observations overexpression of SAF-B1 results in a mild repression of HNF-4α-induced transcription.Στην παρούσα εργασία μελετήθηκε ο βιολογικός ρόλος και οι μοριακές αλληλεπιδράσεις μιας νέας οικογένειας πρωτεϊνικών κινασών που φωσφορυλιώνουν μία ή περισσότερες σερίνες σε διαδοχικές ακολουθίες αργινίνης-σερίνης (RS), και ιδιαίτερα ενός μέλους της οικογένειας, της SRPK1a. Η SRPK1a αποτελεί προϊόν εναλλακτικού ματίσματος του γονιδίου του πρώτου μέλους της οικογένειας, της SRPK1, και περιέχει μία επιπλέον αλλλουχία 171 αμινοξέων στο N-τελικό της άκρο. Σ’ αυτήν την αλληλουχία περιέχεται και το LXXLL μοτίβο, το οποίο έχει θεωρηθεί υπέυθυνο για τη δέσμευση πρωτεϊνών σε πυρηνικούς υποδοχείς. Προκειμένου να βρούμε πρωτεΐνες που αλληλεπιδρούν εξειδικευμένα με την SRPK1a, χρησιμοποιήσαμε το σύστημα των δύο υβριδίων στη ζύμη, επιλέγοντας ως δόλωμα (bait) τo επιπλέον τμήμα των 171 αμινοξέων της SRPK1a. Δύο από τους κλώνους που έδωσαν αλληλεπίδραση ταυτοποιήθηκαν ως τμήματα των γονιδίων που κωδικοποιούν την πρωτεΐνη του πυρηνικού ικριώματος SAF-B1 (Scaffold Attachment Factor B1) και την ογκοκατασταλτική πρωτεΐνη p53 αντίστοιχα. Οι αλληλεπιδράσεις αυτές επιβεβαιώθηκαν με in vitro πειράματα συγκατακρήμνισης και in vivo δοκιμασίες συνανοσοκατακρήμνισης (στην περίπωση του SAF-B1). Επειδή καμία από τις δύο πρωτεΐνες δεν αποτελεί υπόστρωμα της κινάσης αναζητήθηκε κάποια πρωτεΐνη σύνδεσμος. Η έρευνα μας οδήγησε στην πρωτεΐνη P2P-R, η οποία περιέχει RS διπεπτίδια και η οποία ήταν γνωστό από τη βιβλιογραφία ότι δεσμεύει την p53. H RS περιοχή της πρωτεΐνης αυτής βρέθηκε να φωσφορυλιώνεται από την SRPK1a αλλά και να δεσμεύει εξίσου την κινάση. Παράλληλα, η πρωτεΐνη P2P-R βρέθηκε να αλληλεπιδρά, τόσο in vitro όσο και in vivo και με τον SAF-B1, ενώ δοκιμασίες κλασμάτωσης έδειξαν ότι οι δύο αυτές πρωτεΐνες συνεντοπίζονται στο κλάσμα της πυρηνικής μήτρας. Αφού επιβεβαιώθηκαν με πειράματα ανάλυσης της μεταγραφικής ενεργότητας τα βιβλιογραφικά δεδομένα, ότι δηλαδή ο SAF-B1 αναστέλλει την μεταγραφή που εξαρτάται από τον υποδοχέα των οιστρογόνων (ERα), καταδείχθηκε με την ίδια τεχνική ότι και η πρωτεΐνη P2P-R έχει παρόμοια ανασταλτική δράση και μάλιστα ότι αυτή εντοπίζεται στο C-τελικό τμήμα της πρωτεΐνης (αμινοξέα 1314-1516), που όμως δεν περιέχει την RS ακολουθία. Με πειράματα συνανοσοκατακρήμνισης βρέθηκε ότι οι πρωτεΐνες SAF-B1 και P2P-R αλληλεπιδρούν με ενεργοποιητές του μεταγραφικού συμπλόκου του υποδοχέα των οιστρογόνων όπως είναι ο παράγοντας SRC-1 και η μεθυλοτρανσφεράση της αργινίνης CARM1. Παράλληλα, έγινε φανερό ότι ένα μέρος του ERα δεσμεύεται στο κλάσμα της πυρηνικής μήτρας με τη προσθήκη στα κύτταρα του συνδέτη του, της οιστραδιόλης, ή του αγωνιστή της tamoxifen. Η αλλαγή αυτή στην υποπυρηνική εντόπιση του ERα συνοδεύεται και με μία ανάλογη αύξηση δραστικότητας SR κινάσης στο κλάσμα της πυρηνικής μήτρας. Το γεγονός αυτό, σε συνδυασμό με την παρατήρηση ότι επιμόλυνση των κυττάρων με SRPK1a επάγει τη μεταγραφή που ελέγχεται από τον ERα, μας οδήγησε στην πεποίθηση ότι η SRPK1a ρυθμίζει τη συγκρότηση μεγάλων μεταγραφικών συμπλόκων που ελέγχουν την μεταγραφή που εξαρτάται από τον υποδοχέα των οιστρογόνων και τα οποία οργανώνονται σε συνεργασία με στοιχεία της πυρηνικής μήτρας. Επεκτείνοντας την μελέτη μας, βρέθηκε με πειράματα ανάλυσης της μεταγραφικής ενεργότητας ότι η SRPK1a προκαλεί αύξηση και στα επίπεδα μεταγραφής που εξαρτώνται από τον πυρηνικό υποδοχέα HΝF-4α (Hepatic Nuclear Factor-4α). Κυριότερος συνενεργοποιητής του HΝF-4α είναι ο παράγοντας PGC-1 (PPAR γ Coactivator 1), που περιέχει έναν σημαντικό αριθμό RS διπεπτιδίων και βρέθηκε ότι αποτελεί υπόστρωμα κυρίως για την SRPK1a και σε μικρότερη έκταση για την SRPK1. Επιπλέον, δείθηκε ότι η SRPK1a δεσμεύεται στην RS περιοχή του PGC-1 αλλά και στον HNF-4α, πιθανότατα μέσω του LXXLL μοτίβου που περιέχει. Αφαίρεση της RS ακολουθίας από τον PGC-1 είχε ως αποτέλεσμα την μείωση της επαγωγής της μεταγραφικής ενεργότητας του ενδογενούς HNF-4α περίπου κατά 50%. Η παρατήρηση όμως ότι η επαγωγή της μεταγραφής από την SRPK1a εξακολουθεί να υφίσταται ακόμα και αν στα κύτταρα προστεθεί PGC-1-ΔRS, που δεν περιέχει την RS ακολουθία, μας οδηγεί στη σκέψη ότι πιθανόν και άλλα πρωτεϊνικά μόρια που φέρουν RS αλληλουχίες και αποτελούν υποστρώματα της SRPK1a να εμπλέκονται στη μεταγραφή που εξαρτάται από τον HNF-4α. Τέλος, σε αντίθεση με τον υποδοχέα των οιστρογόνων, ο HNF-4α κατανέμεται σχεδόν αποκλειστικά στο κλάσμα του νουκλεοπλάσματος και δεν φαίνεται να συγκροτεί κάποιο μεταγραφικό σύμπλοκο το οποίο να προσδένεται στην πυρηνική μήτρα. Σε συμφωνία με αυτήν την παρατήρηση, υπερέκφραση του SAF-B1 είχε ως αποτέλεσμα μια μικρή μείωση (~30%) στα επίπεδα μεταγραφής που εξαρτάται από τον HNF-4α

eIF2 alpha Kinase PKR Modulates the Hypoxic Response by Stat3-Dependent Transcriptional Suppression of HIF-1 alpha

Hypoxia within the tumor microenvironment promotes angiogenesis, metabolic reprogramming, and tumor progression. In addition to activating hypoxia-inducible factor-1 alpha (HIF-1 alpha), cells also respond to hypoxia by globally inhibiting protein synthesis via serine 51 phosphorylation of translation eukaryotic initiation factor 2 alpha (eIF2 alpha). In this study, we investigated potential roles for stress-activated eIF2 alpha kinases in regulation of HIF-1 alpha. Our investigations revealed that the double-stranded RNA-dependent protein kinase R (PKR) plays a significant role in suppressing HIF-1 alpha expression, acting specifically at the level of transcription. HIF-1 alpha transcriptional repression by PKR was sufficient to impair the hypoxia-induced accumulation of HIF-1 alpha and transcriptional induction of HIF-1 alpha-dependent target genes. Inhibition of HIF-1A transcription by PKR was independent of eIF2 alpha phosphorylation but dependent on inhibition of the signal transducer and activator of transcription 3 (Stat3). Furthermore, HIF-1A repression required the T-cell protein tyrosine phosphatase, which acts downstream of PKR, to suppress Stat3. Our findings reveal a novel tumor suppressor function for PKR, which inhibits HIF-1 alpha expression through Stat3 but is independent of eIF2 alpha phosphorylation. Cancer Res; 70(20); 7820-9. (C) 2010 AACR

Comparison of SARS-CoV-2 indirect and direct RT-qPCR detection methods

Abstract Background Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. Methods Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. Results Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/μl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. Conclusions With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application.

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human β-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2

SAFB1 interacts with and suppresses the transcriptional activity of p53

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity. Structured summary: p53 physically interacts with SRPK1a:shown by two hybrid (view interaction) p53 physically interacts with SRPK1a:shown by pull down (view interaction) p53 physically interacts with SRPK1a:shown by anti bait coimmunoprecipitation (view interaction) p53 physically interacts with SRPK1a:shown by anti tag coimmunoprecipitation (view interaction) SAFB1 physically interacts with p53:shown by pull down (view interactions 1, 2) SAFB1 physically interacts with p53:shown by anti bait coimmunoprecipitation (view interactions 1, 2) SAFB1 and p53 colocalize:shown by fluorescence microscopy (view interaction) SAFB2 physically interacts with p53:shown by pull down (view interaction) (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved