1,720,987 research outputs found
Concepts in sumoylation: a decade on
No full textA decade has passed since SUMO (small ubiquitin-related modifier) was discovered to be a reversible post-translational protein modifier. During this time many enzymes that participate in regulated SUMO-conjugation and -deconjugation pathways have been identified and characterized. In parallel, the search for SUMO substrates has produced a long list of targets, which appear to be involved in most cellular functions. Sumoylation is a highly dynamic process and its outcomes are extremely diverse, ranging from changes in localization to altered activity and, in some cases, stability of the modified protein. At first glance, these effects have nothing in common; however, it seems that they all result from changes in the molecular interactions of the sumoylated proteins
The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus
No full textThe intracellular prolyl peptidase DPP9 is implied to be involved in various cellular pathways including amino acid recycling, antigen maturation, cellular homeostasis, and viability. Interestingly, the major RNA transcript of DPP9 contains two possible translation initiation sites, which could potentially generate a longer (892 aa) and a shorter version (863 aa) of DPP9. Although the endogenous expression of the shorter DPP9 form has been previously verified, it is unknown whether the longer version is expressed, and what is its biological significance. By developing specific antibodies against the amino-terminal extension of the putative DPP9-long form, we demonstrate for the first time the endogenous expression of this longer isoform within cells. Furthermore, we show that DPP9-long represents a significant fraction of total DPP9 in cells, under steady-state conditions. Using biochemical cell fractionation assays in combination with immunofluorescence studies, we find the two isoforms localize to separate subcellular compartments. Whereas DPP9-short is present in the cytosol, DPP9-long localizes preferentially to the nucleus. This differential localization is attributed to a classical monopartite nuclear localization signal (K(K/R)X(K/R)) in the N-terminal extension of DPP9-long. Furthermore, we detect prolyl peptidase activity in nuclear fractions, which can be inhibited by specific DPP8/9 inhibitors. In conclusion, a considerable fraction of DPP9, which was previously considered as a purely cytosolic peptidase, localizes to the nucleus and is active there, raising the intriguing possibility that the longer DPP9 isoform may regulate the activity or stability of nuclear proteins, such as transcription factors
A Novel SUMO1-specific Interacting Motif in Dipeptidyl Peptidase 9 (DPP9) That Is Important for Enzymatic Regulation
No full textSumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu(67) of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9.Deutsche Forschungsgemeinschaft [2234/1-1]; European Union NoE Rubico
The SUMO1-E67 interacting loop peptide is an allosteric inhibitor of the dipeptidyl peptidases 8 and 9.
Get PDFThe intracellular peptidases dipeptidyl peptidase (DPP) 8 and DPP9 are involved in multiple cellular pathways including antigen maturation, cellular homeostasis, energy metabolism, and cell viability. Previously we showed that the small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP9, leading to allosteric activation of the peptidase. Here we demonstrate that the E67-interacting loop (EIL) peptide, which corresponds to the interaction surface of SUMO1 with DPP9, acts as a noncompetitive inhibitor of DPP9. Moreover, by analyzing the sensitivity of DPP9 arm mutants to the EIL peptide, we mapped specific residues in the arm that are important for inhibition by the EIL, suggesting that the peptide acts as an allosteric inhibitor of DPP9. By modifying the EIL peptide, we constructed peptide variants with more than a 1,000-fold selectivity toward DPP8 (147 nm) and DPP9 (170 nm) over DPPIV (200 μm). Furthermore, application of these peptides to cells leads to a clear inhibition of cellular prolyl peptidase activity. Importantly, in line with previous publications, inhibition of DPP9 with these novel allosteric peptide inhibitors leads to an increase in EGF-mediated phosphorylation of Akt. This work highlights the potential use of peptides that mimic interaction surfaces for modulating enzyme activity
The lincRNA Pantr1 is a FOXG1 target gene conferring site-specific chromatin binding of FOXG1
No full textDerailed gene expression programs within the developing nervous system, encompassing both transcriptional and post-transcriptional processes, can cause diverse neurodevelopmental diseases (NDD). The NDD FOXG1-syndrome lacks full understanding of the mechanistic role of its eponymous gene product. While it is known that FOXG1 acts in part at the chromatin by binding to regulative regions, it is unclear what factors control its presence at specific sites. Long non-coding RNAs (lncRNAs) can mediate site-directed transcription factor binding, but their potential role in FOXG1-syndrome has not been described. Here, we show that FOXG1 localisation is regulated at selected loci through the lncRNA Pantr1. We identified FOXG1 as an upstream transcriptional activator of Pantr1 in human and mice. Further, we discovered that FOXG1 has the ability to associate with RNAs. Both transcriptional regulation of Pantr1 by FOXG1 and binding of both partners build up a regulative network that impacts the localisation of FOXG1 at selected genomic loci. Specifically, Pantr1 facilitates cooperative presence of FOXG1/NEUROD1 at specific sites, and Pantr1 reduction leads to redistribution of FOXG1 to comparably more generic binding sites. The rescue of impaired dendritic outgrowth upon FOXG1 reduction by simultaneous overexpression of Pantr1 underlines the importance of the FOXG1/Pantr1 regulative network
Dipeptidyl peptidase 9 is a positive regulator of the ufolded protein response
No full textDipeptidyl peptidase 9 (DPP9) is an intracellular amino dipeptidase of the DPPIV serine protease family. It is involved in the immune response, neonatal survival, and DNA repair, while dysregulation of DPP9 has been linked to cancer. In a screen for novel DPP9 substrates, the intracellular endoplasmic reticulum (ER) membrane-bound protein ATF6 (Activating Transcription Factor 6) was identified as a putative DPP9 target. ATF6 is among the three sensors of the intracellular signaling pathway termed the unfolded protein response (UPR), which is activated upon accumulation of misfolded proteins in the ER. In the presence of ER-stress, ATF6 undergoes processing to release the N-terminal domain of ATF6 (ATF6N), which acts as a transcription factor to induce gene expression of UPR target genes. This study focuses on ATF6 as a substrate of DPP9, and investigated whether DPP9 plays a role in the UPR. Analysis of mRNA and protein levels of key players involved in the UPR, demonstrate that depletion of DPP9 in HeLa and HEK 293 cells leads to defects in UPR signaling affecting both early and late UPR responses to various types of ER stress. These effects can be reversed by the re-expression of DPP9. Further, the interaction between DPP9 and ATF6 was characterized in-vitro using ATF6 N-terminal peptides and recombinant DPP9; as well as in cells using immuno-precipitation and NanoBRET assays. Critical residues of ATF6 for their interaction with DPP9 were mapped, and stable cell lines were generated for the doxycycline-induced expression of ATF6N and ATF6N mutated in the DPP9 cleavage site. In vitro assays with recombinant DPP9 and ATF6 N-terminal peptides demonstrate cleavage of ATF6N at Proline-14. Importantly, in contrast to wild-type ATF6N, cells expressing ATF6N P14G showed defects in UPR signaling similar to the defects observed in the DPP9 knock-out cells. This suggests that DPP9 promotes the UPR signaling, and that this effect requires ATF6. Consistently, depletion of DPP9 in HEK 293 and HeLa cells, resulted in a hyper-sensitivity to induced ER stress. Importantly, also J558L multiple myeloma cells show a higher sensitivity to ER stress following treatment with 1G244, an inhibitor of DPP8/9. In summary, this highlights a novel role for DPP9 as a positive regulator of the UPR by cleaving ATF6, and expands our understanding of mechanisms regulating cellular responses to stress
EPITHELIAL CELL MOVEMENT AND NUCLEAR AND CYTOPLASMIC FORMS OF THE ESSENTIAL PROTEASE DIPEPTIDYL PEPTIDASE 9 (DPP9)
No full textStructures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer
No full textDipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 angstrom) and DPP9 (3.0 angstrom) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one ss-propeller and a/ss hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an alpha-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets
Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP)
No full text- …
