Project description:SDS22 forms an inactive complex with nascent protein phosphatase-1 (PP1) and Inhibitor-3 (I3). SDS22:PP1:I3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 prevents the aggregation of nascent PP1. In the absence of SDS22, PP1 was gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. A human patient with an unstable SDS22 mutant also expressed reduced levels of PP1 and suffered from neurodevelopmental retardation. We furthermore found that SDS22 directly binds to I3 and that this is essential for the stable assembly of SDS22:PP1:I3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled I3-binding site co-transfered with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, by its ability to bind to both PP1 and I3, integrates the major steps of PP1 holoenzyme assembly.
Project description:The goal of the project was to check the differential effect on transcription rate of a sudden degron-induced depletion of Hpr1 protein and its comparison towards the effect of a constitutive absence of Hpr1 protein in a deletion strain. Cells were grown in YP-Raffinose + 0.1 mM CuSO4 at 26ºC to 0.5 OD600. At that cells were spun-down and changed to YP-Galactose without CuSO4 for 30 min at 26ºC. Then cells were shifted to 37ºC to induce degron-mediated proteolysis and recovered after 30 min. Samples include wt, hpr1delta and hpr1-degron after 0 or 30 min growth at 30ºC.
Project description:The deletion of the protein phosphatase-1 (PP1) regulator NIPP1 is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1. In total 16 samples were processed. Four different cell lines were analysed: HTO_parental, HTO_NIPP1wt, HTO_NIPP1m (= alias NIPP1-Pm) and HTO_NIPP1-Pa. For each cell line 4 replicates were obtained. The HTO_parental cell line is the control cell line. For the HTO_NIPP1wt and the HTO_NIPP1-Pa the 4 replicates were obtained from two replicates of two different transgenic cell lines expressing FlagNIPP1wt (cell line wt n°1 and 2) and FlagNIPP1-Pa (cell line n°1 and 2), respectively. Each cell line was derived from the same parental control cell line.
Project description:Background: While heart transplantation is the standard treatment for heart failure, both acute and chronic transplant rejection frequently occur. Since the modulation of protein phosphatase PP2A activity is critical for tissue and organ homeostasis under physiological and pathophysiological conditions, PP2A may also affect the ability to tolerate transplanted organs. Here we demonstrate that administration of a novel class of small molecule activators of PP2A (SMAPs) prolonged cardiac allograft survival in a mouse heterotopic heart transplantation model. Mechanistically, SMAPs effectively suppressed the inflammatory immune response while increasing Treg population in the allografts, findings corroborated by functional analysis of RNAseq data derived from Tregs of treated splenic tissue. Importantly, SMAPs further prolonged CTLA4-Ig (an immunosuppressive agent utilized in organ transplantation)-induced cardiac rejection tolerance and extended allograft survival. SMAPs also strongly mitigated cardiac allograft vasculopathy as evidenced by a marked reduction of neointimal hyperplasia and smooth muscle cell proliferation. Mechanistic studies implicate SMAPs elicited suppression of MEK/ERK pathways as a unifying mechanism for the aforementioned effects in Tregs and SMCs. These findings highlight the potential of PP2A activation in improving alloengraftment in heart transplantation and add knowledge to the phosphatase driven regulation of the immune system in the context of organ transplantation.
