Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity

<p>Abstract</p> <p>Background</p> <p>Retrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks.</p> <p>Results</p> <p>Here we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12^thand 59^thamino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio <it>in vivo</it>, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen.</p> <p>Conclusions</p> <p>Together, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitt's lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS) warrants further investigation into its role in disease development.</p

Determination of Ras-GTP and Ras-GDP in patients with acute myelogenous leukemia (AML), myeloproliferative syndrome (MPS), juvenile myelomonocytic leukemia (JMML), acute lymphocytic leukemia (ALL), and malignant lymphoma: assessment of mutational and indirect activation

The 21-kD protein Ras of the low-molecular-weight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a ‘molecular switch’ in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of N- and K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through “upstream” or “downstream” mechanisms

Mammalian mitochondrial nitric oxide synthase: Characterization of a novel candidate

AbstractRecently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development

Rap Signaling in Normal Lymphocyte Development and Leukemia Genesis

Although Rap GTPases of the Ras family remained enigmatic for years, extensive studies in this decade have revealed diverse functions of Rap signaling in the control of cell proliferation, differentiation, survival, adhesion, and movement. With the use of gene-engineered mice, we have uncovered essential roles of endogenous Rap signaling in normal lymphocyte development of both T- and B-lineage cells. Deregulation of Rap signaling, on the other hand, results in the development of characteristic leukemia in manners highly dependent on the contexts of cell lineages. These results highlight crucial roles of Rap signaling in the physiology and pathology of lymphocyte development

PBX1 is dispensable for neural commitment of RA-treated murine ES cells

Experimentation with PBX1 knockout mice has shown that PBX1 is necessary for early embryogenesis. Despite broad insight into PBX1 function, little is known about the underlying target gene regulation. Utilizing the Cre–loxP system, we targeted a functionally important part of the homeodomain of PBX1 through homozygous deletion of exon-6 and flanking intronic regions leading to exon 7 skipping in embryonic stem (ES) cells. We induced in vitro differentiation of wild-type and PBX1 mutant ES cells by aggregation and retinoic acid (RA) treatment and compared their profiles of gene expression at the ninth day post-reattachment to adhesive media. Our results indicate that PBX1 interactions with HOX proteins and DNA are dispensable for RA-induced ability of ES to express neural genes and point to a possible involvement of PBX1 in the regulation of imprinted genes

HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer

FPGA-accelererad paketfångst med eBPF : Prestandaöverväganden vid användning av SoC FPGA acceleratorer för paketering.

With the rise of the Internet of Things and the proliferation of embedded devices equipped with an accelerator arose a need for efficient resource utilization. Hardware acceleration is a complex topic that requires specialized domain knowledge about the platform and different trade-offs that have to be made, especially in the area of power consumption. Efficient work offloading strives to reduce or at least maintain the total power consumption of the system. Offloading packet capturing is usually done in more powerful devices, hence scarce research is present concerning network packet acceleration in embedded devices. The thesis focuses on accelerating networking packets utilizing a Field Programmable Gate Array in an embedded Linux System. The solution is based on a custom Linux distribution assembled using the Buildroot tool, specially configured and patched Linux kernel, uboot bootloader, and the programmable logic for packet acceleration. The system is evaluated on a De0-Nano System on Chip development board through modifications to burst lengths, packet sizes, and programmable logic clock frequency. Metrics include packet capturing time, time per packet, and consumed power. Finally, the results are contrasted with baseline embedded Linux packet processing by inspection of a packet’s path through the kernel. Collected results provide a deeper understanding of the packet acceleration problem in embedded devices and the resultant system gives a solid starting point for possible extensions such as packet filtering. Key findings include an improvement in packet processing speed as the clock frequency and burst length are increased while maintaining power consumption. Additionally, the solution performs better when the packet sizes are above 64 bytes as the overhead of additional logic necessary for their processing is compensated. The project is also found to be significantly faster than regular in kernel processing with the caveat of providing just packet capturing whereas Linux contains a full network stack.I och med uppkomsten av sakernas internet och spridningen av inbyggda enheter som är utrustade med en accelerator har det uppstått ett behov av effektivt resursutnyttjande. Hårdvaruacceleration är ett komplext ämne som kräver specialiserad domänkunskap om plattformen och olika avvägningar som måste göras, särskilt när det gäller energiförbrukning. Effektiv arbetsavlastning strävar efter att minska eller åtminstone bibehålla systemets totala energiförbrukning. Avlastning av paketering sker vanligtvis i kraftfullare enheter, och därför finns det knappt någon forskning om nätverksacceleration av paket i inbyggda enheter. Avhandlingen är inriktad på att påskynda nätverkspaket med hjälp av en Field Programmable Gate Array i ett inbäddat Linuxsystem. Lösningen bygger på en anpassad Linuxdistribution som sammanställts med hjälp av verktyget Buildroot, en särskilt konfigurerad och patchad Linuxkärna, uboot bootloader och den programmerbara logiken för paketacceleration. Systemet utvärderas på ett De0-Nano System on Chip-utvecklingskort genom ändringar av burstlängder, paketstorlekar och den programmerbara logikens klockfrekvens. Metrikerna omfattar tid för paketering, tid per paket och förbrukad effekt. Slutligen jämförs resultaten med grundläggande inbäddad Linux-paketbehandling genom inspektion av paketens väg genom kärnan. De samlade resultaten ger en djupare förståelse för problemet med paketacceleration i inbyggda enheter och det resulterande systemet ger en solid utgångspunkt för möjliga utvidgningar, t.ex. paketfiltrering. Bland de viktigaste resultaten kan nämnas en förbättring av hastigheten i paketbehandlingen när klockfrekvensen och burstlängden ökas samtidigt som strömförbrukningen bibehålls. Dessutom fungerar lösningen bättre när paketstorleken är större än 64 bytes eftersom den extra logik som krävs för att behandla paketen kompenseras. Projektet har också visat sig vara betydligt snabbare än vanlig kärnbearbetning, med den reservationen att det bara tillhandahåller paketupptagning, medan Linux innehåller en fullständig nätverksstack.Rozwój Internetu Rzeczy i ąrosnca śćpopularno systemów wbudowanych ąposiadajcych wbudowany akcelerator ęsprztowy łsprawiy, że łwzrosa potrzeba na ich efektywne wykorzytanie. Akceleracja ęsprztowa jest ądziedzin nauki, która wymaga specjalistycznej wiedzy na temat platformy na której ma ćoperowa oraz wymaga śznajomoci potencjalnych komplikacji które ęsi z ąni ążąwi. Efektywna akceleracja ma na celu ęredukcj żzuycia energii, a przynajmnniej jej utrzymanie na dotychczasowym poziomie. Tematyka ta jest śćdo uboga pod ąktem ędostpnej literatury, żgdy zazwyczaj akceleratory stosowane do sieciowych ąńrozwiza ąs żuywane w ąrozwizaniach serwerowych gdzie ęąwystpuj innego rodzaju problemy. W pracy wykorzystany jest akcelerator Field Programmable Gate Array który jest ęśączci łpytki deweloperskiej De0-Nano System on Chip, gdzie łdziaa łąwspópracujc z wbudowanym systemem Linux, do którego przygotowania wykorzystano ęnarzdzie Buildroot. Na ńkocowe ąrozwizanie ponadto łskada ęsi łpoatane ąjdro Linuxa, bootloader uboot oraz programowalna logika ąrealizujca przechwytywanie pakietów sieciowych. ąRozwizanie poddane jest testom, w których parametry odpowiedzialne za łśćdugo transakcji typu burst, rozmiaru pakietu oraz ęśczstotliwoci zegara ąs poddawane modyfikacjom. Wyniki ąs przedstawione za ąpomoc czasu przetwarzania pakietu, czasu per pakiet oraz żzuycia mocy. Do oceny śefektywnoci ąrozwizania łżłposuyo żtake porównanie z czasem procesowania pakietu w niezmodyfikowanym systemie Linux Na podstawie eksperymentów dokonanych w pracy ęwysunite ąs ęąnastpujce wnioski: wraz ze wzrostem ęśczstotliwoci zegara oraz łśdugoci transakcji burst, czas procesowania pakietów maleje a żzuycie ąprdu pozostaje na dotychczasowym poziomie. Pakiety o rozmiarze ąprzekraczajcym 64 bajty ąs procesowane wydajniej w dostarczonym ąrozwizaniu poprzez ękompensacj dodatkowego łnakadu czasu narzuconego przez ęlogik ąąązarzdzajc przetwarzaniem. System porównano żtake do łzwykego przetwarzania pakietów ąodbywajcego ęsi w systemie Linux które łokazao ęsi zdecydowanie wolniejsze z żzastrzeeniem, żi ów system dokonuje łpenego przetworzenia pakietów a ąrozwizanie w pracy jedynie ich przechwytywania. Projekt stanowi ępodstaw do ewentualnych ńrozszerze, na łprzykad filtrowania pakietów. Wnioski ęwysunite łżąsu łępogbieniu wiedzy w domenie sieci wbudowanych systemów Linux oraz ęsprztowej akceleracji