1H, 13C and 15N NMR chemical shift assignments of cAMP-regulated phosphoprotein-19 and -16 (ARPP-19 and ARPP-16)

Protein Phosphatase 2A, PP2A, the principal Serine/threonine phosphatase, has major roles in broad range of signaling pathways that include regulation of cell cycle, cell proliferation and neuronal signaling. The loss of function of PP2A is linked with many human diseases, like cancer and neurodegenerative disorders. Protein phosphatase 2A (PP2A) functions as tumor suppressor and its tumor suppressor activity is inhibited by the overexpression of PP2A inhibitor proteins in most of the cancers. ARPP-19/ARPP-16 has been identified as one of the potential PP2A inhibitor proteins. Here, we report the resonance assignment of backbone 1H, 13C and 15N atoms of human ARPP-19 and ARPP-16 proteins. These chemical shift values can provide valuable information for the further study of the dynamics and interaction of ARPP-proteins to PP2A using NMR spectroscopy.</p

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

<p>Abstract</p> <p>Background</p> <p>Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in <it>Escherichia coli </it>in simple shaken cultures.</p> <p>Results</p> <p>The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD₆₀₀~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l^-1cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.</p> <p>Conclusions</p> <p>We have demonstrated that by applying the novel EnBase^®Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.</p

The Interaction Mechanism of Intrinsically Disordered PP2A Inhibitor Proteins ARPP-16 and ARPP-19 With PP2A

Protein phosphatase 2A (PP2A) activity is critical for maintaining normal physiological cellular functions. PP2A is inhibited by endogenous inhibitor proteins in several pathological conditions including cancer. A PP2A inhibitor protein, ARPP-19, has recently been connected to several human cancer types. Accordingly, the knowledge about ARPP-19-PP2A inhibition mechanism is crucial for the understanding the disease development and the therapeutic targeting of ARPP-19-PP2A. Here, we show the first structural characterization of ARPP-19, and its splice variant ARPP-16 using NMR spectroscopy, and SAXS. The results reveal that both ARPP proteins are intrinsically disordered but contain transient secondary structure elements. The interaction mechanism of ARPP-16/19 with PP2A was investigated using microscale thermophoresis and NMR spectroscopy. Our results suggest that ARPP-PP2A A-subunit interaction is mediated by linear motif and has modest affinity whereas, the interaction of ARPPs with B56-subunit is weak and transient. Like many IDPs, ARPPs are promiscuous binders that transiently interact with PP2A A- and B56 subunits using multiple interaction motifs. In summary, our results provide a good starting point for future studies and development of therapeutics that block ARPP-PP2A interactions

Interaction mechanism of endogenous PP2A inhibitor protein ENSA with PP2A

The vast diversity of protein phosphatase 2A (PP2A) holoenzyme composition ensures its multifaceted role in the regulation of cellular growth and signal transduction. In several pathological conditions, such as cancer, PP2A is inhibited by endogenous inhibitor proteins. Several PP2A inhibitor proteins have been identified, one of which is alpha-endosulfine (ENSA). ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. The role of ENSA in PP2A inhibition is rather well characterized, but knowledge of the mechanism of inhibition is scarce. In this study, we have performed comprehensive structural characterization of ENSA, and its interaction with PP2A A- and various B56-subunit isoforms by combining NMR spectroscopy, small-angle X-ray scattering (SAXS) and interaction assays. The results clearly indicate that ENSA is an intrinsically disordered protein containing three transient alpha-helical structures. ENSA was observed to interact PP2A mainly via A-subunit, as the affinity with the A-subunit is significantly stronger than with any of the B56 subunits. Based on our results, it seems that ENSA follows the dock-and-coalesce mechanism in associating with PP2A A-subunit. Taken together, our results provide an essential structural and molecular framework to understanding molecular bases of ENSA-mediated PP2A inhibition, which is crucial for the development of new therapies for diseases linked to PP2A inhibition

Peroxisomal multifunctional enzyme type 2 (MFE-2):the catalytic domains work as independent units

Abstract Lipids are necessary for living organisms and have various roles as, energy sources, hormone precursors and as components of membrane structures. Fatty acid β-oxidation is a pathway of energy metabolism, in which the fatty acyl-CoAs are degraded in several steps. Peroxisomal β-oxidation systems are found in all eukaryotes studied thus far, but the existence of a mitochondrial system is established in mammals only. Multifunctional enzyme type 2 (MFE-2) has been characterized from various species and is responsible for catalyzing the second and third steps in the R-specific peroxisomal β-oxidation pathway. MFE-2 accepts a wide range of substrates and displays great variation in domain organization and overall molecular mass. The crystal structures of individual domains of MFE-2 from several species have been determined previously. In this study, the structural knowledge of MFE-2 is further extended from the domain level to the assembly of the full-length enzyme. The crystal structure of Drosophila melanogaster MFE-2 (DmMFE-2) was solved at 2.15 Å resolution. The enzyme is a homodimer with 3R-hydroxyacyl-CoA dehydrogenase and 2E-enoyl-CoA hydratase 2 activities residing on each of the polypeptide subunits. Kinetic data combined with information from the structure, suggest that the catalytic domains of DmMFE-2 work as separate entities. It also appears that the enzyme does not assemble into dimers in vitro when the catalytic subunits are introduced in a solution as stand-alone proteins. The data were confirmed by two different methods, static light scattering and small-angle X-ray scattering. However, the primary use of SAXS was not to monitor the formation of dimers in solution, but instead it was used for structure determination of the human MFE-2. During the process, the structural information from DmMFE-2 was used as a scaffold for the human homolog. After collecting a wide range of SAXS data and numerous calculations, a plausible low resolution solution structure of human MFE-2 was obtained. The model reveals the overall assembly of the enzyme and the locations of the C-terminal SCP-2L domains (an unspecific lipid carrier), thus enabling further hypotheses regarding the possible role of the SCP-2L domain in the enzymatic reaction.Tiivistelmä Lipidit eli rasva-aineet ovat välttämättömiä eliöille ja niillä on lukemattomia rooleja mm. energianlähteinä, kalvojen rakenteina ja hormonien esiasteina. Rasvahappoja hajotetaan monivaiheisella metaboliareitillä, jota kutsutaan β-oksidaatioksi. Peroksisomaalinen rasvojen hajotusreitti on löydetty kaikista tähän asti tutkituista aitotumallisista, mutta mitokondrioissa tapahtuva rasvojen β-oksidaatio on löydetty vain nisäkkäiltä. Peroksisomaalinen monitoiminen entsyymi tyyppi 2 (MFE-2) katalysoi toisen ja kolmannen reaktion R-spesifisellä rasvahappojen hajotusreitillä ja se on karakterisoitu useilta eri lajeilta. MFE-2 muodostaa lajista riippuen hyvin erilaisia ja molekyylimassaltaan erikokoisia alayksikköyhdistelmiä, jotka pystyvät katalysoimaan erityyppisten substraattien hapetuksen. Eri lajien MFE-2:n yksittäisten alayksiköiden kiderakenteet ovat olleet tunnettuja jo vuosia. Tässä tutkimuksessa rakennetietämys laajenee MFE-2:n osalta alayksikkötasolta kokopitkän entsyymin tasolle, sillä banaanikärpäsen MFE-2:n (DmMFE-2) kiderakenne selvitettiin 2.15 ångströmin erotuskyvyllä. Tämä homodimeerinen entsyymi kantaa samassa polypeptidissä sekä 3R-hydroksiasyyli-KoA-dehydrogenaasi, että 2E-enoyyli-KoA-hydrataasi 2 -aktiivisuuksia. Kiderakenteen ja reaktiokinetiikan perusteella tehtiin johtopäätös, jonka mukaan DmMFE-2:n alayksiköt toimivat itsenäisinä kokonaisuuksinaan. Staattisen valonsironnan (SLS) ja röntgenpienkulmasirontamittauksien (SAXS) perusteella MFE-2:n erillisinä tuotetut alayksiköt eivät muodosta liuoksessa spontaanisti kokopitkän MFE-2:n kaltaisia oligomeerejä. Ihmisen MFE-2:n alhaisen erotuskyvyn malli määritettiin röntgenpienkulmasirontatekniikan avulla. Tässä prosessissa käytettiin hyväksi banaanikärpäsen entsyymin tarjoamaa rakennetietoa, jonka perusteella rakennettiin ensin runko ihmisen MFE-2:lle. Monivaiheisen prosessin jälkeen saatiin lopulta laskettua vakuuttava malli, joka paljastaa ensimmäistä kertaa ihmisen kokopitkän MFE-2:n rakenteen ja antaa mahdollisuuden tehdä alustavia johtopäätöksiä karboksiterminaalisen lipidejä epäspesifisesti sitovan alayksikön (SCP-2L) biologisesta roolista osana tätä monitoimista entsyymiä

1H, 13C and 15N NMR chemical shift assignments of cAMP-regulated phosphoprotein-19 and -16 (ARPP-19 and ARPP-16)

Protein Phosphatase 2A, PP2A, the principal Serine/threonine phosphatase, has major roles in broad range of signaling pathways that include regulation of cell cycle, cell proliferation and neuronal signaling. The loss of function of PP2A is linked with many human diseases, like cancer and neurodegenerative disorders. Protein phosphatase 2A (PP2A) functions as tumor suppressor and its tumor suppressor activity is inhibited by the overexpression of PP2A inhibitor proteins in most of the cancers. ARPP-19/ARPP-16 has been identified as one of the potential PP2A inhibitor proteins. Here, we report the resonance assignment of backbone 1H, 13C and 15N atoms of human ARPP-19 and ARPP-16 proteins. These chemical shift values can provide valuable information for the further study of the dynamics and interaction of ARPP-proteins to PP2A using NMR spectroscopy.peerReviewe

Interaction mechanism of endogenous PP2A inhibitor protein ENSA with PP2A

The vast diversity of protein phosphatase 2A (PP2A) holoenzyme composition ensures its multi-faceted role in the regulation of cellular growth and signal transduction. In several pathological conditions, such as cancer, PP2A is inhibited by endogenous inhibitor proteins. Several PP2A inhibitor proteins have been identified, one of which is α-endosulfine (ENSA). ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. The role of ENSA in PP2A inhibition is rather well characterized, but knowledge of the mechanism of inhibition is scarce. In this study, we have performed comprehensive structural characterization of ENSA, and its interaction with PP2A A- and various B56-subunit isoforms by combining NMR spectroscopy, SAXS and interaction assays. The results clearly indicate that ENSA is an intrinsically disordered protein containing three transient α-helical structures. ENSA was observed to interact PP2A mainly via A-subunit, as the affinity with the A-subunit is significantly stronger than with any of the B56-subunits. Based on our results, it seems that ENSA follows the dock-and-coalesce mechanism in associating with PP2A A-subunit. Taken together, our results provide an essential structural and molecular framework to understanding molecular bases of ENSA mediated PP2A inhibition, which is crucial for the development of new therapies for diseases linked to PP2A inhibition.peerReviewe

The Interaction Mechanism of Intrinsically Disordered PP2A Inhibitor Proteins ARPP-16 and ARPP-19 With PP2A

Protein phosphatase 2A (PP2A) activity is critical for maintaining normal physiological cellular functions. PP2A is inhibited by endogenous inhibitor proteins in several pathological conditions including cancer. A PP2A inhibitor protein, ARPP-19, has recently been connected to several human cancer types. Accordingly, the knowledge about ARPP-19—PP2A inhibition mechanism is crucial for the understanding the disease development and the therapeutic targeting of ARPP-19—PP2A. Here, we show the first structural characterization of ARPP-19, and its splice variant ARPP-16 using NMR spectroscopy, and SAXS. The results reveal that both ARPP proteins are intrinsically disordered but contain transient secondary structure elements. The interaction mechanism of ARPP-16/19 with PP2A was investigated using microscale thermophoresis and NMR spectroscopy. Our results suggest that ARPP—PP2A A-subunit interaction is mediated by linear motif and has modest affinity whereas, the interaction of ARPPs with B56-subunit is weak and transient. Like many IDPs, ARPPs are promiscuous binders that transiently interact with PP2A A- and B56 subunits using multiple interaction motifs. In summary, our results provide a good starting point for future studies and development of therapeutics that block ARPP-PP2A interactions.peerReviewe

ZnT8 autoantibody epitope specificity and affinity examined with recombinant ZnT8 variant proteins in specific ZnT8R and ZnT8W autoantibody positive type 1 diabetes patients.

Variant specific zinc transporter 8 autoantibodies (ZnT8A) against either arginine (R) or tryptophan (W) at amino acid (aa) position 325 of the zinc transporter 8 (ZnT8) has been identified in T1D patients. Reciprocal cross-over tests revealed differences in half-maximal binding to indicate variable affinity of patient ZnT8 autoantibodies. Insufficient recombinant ZnT8 variant proteins have precluded detailed analyses of ZnT8 autoantibody affinity. The aims in the present study were to 1) generate recombinant ZnT8R- and ZnT8W-aa275-369 proteins 2) test the ZnT8R- and ZnT8W-aa275-369 proteins in reciprocal competitive radiobinding assays (RBA) against ZnT8R- and ZnT8W-aa268-369 labeled with (35) S-methionine, and 3) determine the specificity and affinity of sera specific for either ZnT8 Arginine (R) or ZnT8 Tryptophan (W) autoantibodies in newly diagnosed type 1 diabetes (T1D) patients. The results demonstrate first that it was possible to produce recombinant human MBP-ZnT8aa275-369 protein purified to homogeneity for RBA reciprocal competition experiments. Second, high titer ZnT8WA sera diluted to half maximal binding showed significant specificity for respective variants of either ZnT8R or ZnT8W. Third, ZnT8WA positive sera showed high affinity for ZnT8W compared to ZnT8RA for ZnT8R. These data demonstrate that T1D patients may have single amino acid specific autoantibodies directed against either ZnT8R or ZnT8W and that the autoantibody affinity to the respective variant may be different. Further studies are needed to assess the mechanisms by which variant specific ZnT8A of variable affinity develop and their possible role in the pathogenic process leading to the clinical onset of T1D