RESUMO
PTPRK is a receptor tyrosine phosphatase that is linked to the regulation of growth factor signalling and tumour suppression. It is stabilized at the plasma membrane by trans homophilic interactions upon cell-cell contact. PTPRK regulates cell-cell adhesion but is also reported to regulate numerous cancer-associated signalling pathways. However, the signalling mechanism of PTPRK remains to be determined. Here, we find that PTPRK regulates cell adhesion signalling, suppresses invasion and promotes collective, directed migration in colorectal cancer cells. In vivo, PTPRK supports recovery from inflammation-induced colitis. In addition, we confirm that PTPRK functions as a tumour suppressor in the mouse colon and in colorectal cancer xenografts. PTPRK regulates growth factor and adhesion signalling, and suppresses epithelial to mesenchymal transition (EMT). Contrary to the prevailing notion that PTPRK directly dephosphorylates EGFR, we find that PTPRK regulation of both EGFR and EMT is independent of its catalytic function. This suggests that additional adaptor and scaffold functions are important features of PTPRK signalling.
Assuntos
Transição Epitelial-Mesenquimal , Humanos , Animais , Camundongos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Transdução de Sinais , Adesão Celular/genética , Movimento Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Colite/patologia , Colite/metabolismo , Colite/genética , Colite/induzido quimicamente , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Intestinos/patologiaRESUMO
Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
BACKGROUND: The impact of changes in novel agent (NA) usage on the survival of multiple myeloma (MM) patients in real-world hospital settings is unclear. In New Zealand (NZ) in 2011, frontline bortezomib became available and thalidomide availability was expanded. AIM: This retrospective study analyses the impact these change had on the survival of MM patients treated at a NZ hospital. METHODS: Clinical and overall survival (OS) data were collected on MM patients who were treated at Christchurch Hospital during 2000-2009 (pre-cohort, n = 337) and 2011-2017 (post-cohort, n = 343). Outcomes were compared using pre-cohort data truncated at 2011. RESULTS: Patients in the post-cohort had significant increases (P < 0.001) in not only NA usage (85 vs 55%) and OS (median = 56 vs 44 months) but also the proportion (74 vs 49%) of young patients (age < 70) who received an autologous stem cell transplant (ASCT). Separate analysis of older patients demonstrated that those in the post-cohort had significantly longer OS (median OS 28 vs 17, P < 0.001) although 5-year relative survival remained less than 50%. Separate analysis of young patients demonstrated that those in the post-cohort had significantly increased initial OS with the survival curves converging at 5 years. Although ASCT-treated patients had similar OS in each cohort, their progression-free survival (PFS) was significantly increased in the post-cohort (median 40 vs 20 months, P < 0.0001). CONCLUSION: In the setting of a NZ hospital the increased availability of NA was associated with a significant improvement in both the OS of older patients and the PFS of ASCT patients.
Assuntos
Acessibilidade aos Serviços de Saúde/tendências , Transplante de Células-Tronco Hematopoéticas/tendências , Hospitalização/tendências , Mieloma Múltiplo/epidemiologia , Mieloma Múltiplo/terapia , Intervalo Livre de Progressão , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos Alquilantes/administração & dosagem , Bortezomib/administração & dosagem , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunossupressores/administração & dosagem , Quimioterapia de Indução/métodos , Quimioterapia de Indução/tendências , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Nova Zelândia/epidemiologia , Estudos Retrospectivos , Talidomida/administração & dosagem , Transplante Autólogo/métodos , Transplante Autólogo/tendências , Resultado do TratamentoRESUMO
Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early-onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.
Assuntos
Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Niclosamida/análogos & derivados , Niclosamida/farmacologia , Proteínas Quinases/metabolismo , Descoberta de Drogas , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/enzimologiaRESUMO
The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Receptores de Estrogênio/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Biocatálise , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Camundongos , Modelos Moleculares , Terapia de Alvo Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Idiopathic juvenile osteoporosis (IJO) is a rare condition presenting with vertebral and metaphyseal fractures that affects otherwise healthy prepubertal children. Bone mineral density (BMD) measurements are very low. The primary problem appears to be deficient bone formation, with a failure to accrue bone normally during growth. The onset in childhood suggests IJO is a genetic disorder, and a number of reports indicate that some children carry heterozygous pathogenic variants in genes known to be associated with defective osteoblast function and low bone mass, most commonly LRP5 or PLS3. However, a positive family history is unusual in IJO, suggesting the genetic background can be complex. We describe a young man with classical IJO who was investigated with a bone fragility gene panel and whole genome sequencing. The proband was found to carry four variants in three different genes potentially affecting osteoblast function. From his mother he had inherited mutations in ALPL (p.Asn417Ser) and LRP5 (p.Arg1036Gln), and from his father mutations in LRP5 (p.Asp1551Alsfs*13) and activating transcription factor 4 (ATF4) (p.Leu306Ile). His sister had also inherited the LRP5 (p.Asp1551Alsfs*13) from her father, but not the ATF4 mutation. Their spinal BMD z-scores differed substantially (sister -1.6, father -3.2) pointing to the potential importance of the ATF4 mutation. Activating transcription factor 4 acts downstream from RUNX2 and osterix and plays an important role in osteoblast differentiation and function. This case, together with others recently published, supports the view that IJO can result from clustering of mutations in genes related to osteoblast development and function. Novel genes in these pathways may be involved. Our case also emphasizes the value of detailed study of other family members. After a bone biopsy had excluded a mineralization defect due to hypophosphatasia, the proband was treated with zoledronate infusions with good clinical effect.
RESUMO
Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic activity. Previously we showed that PTPRK promotes cell-cell adhesion by selectively dephosphorylating several cell junction regulators including the protein Afadin (Fearnley et al, 2019). Here, we demonstrate that Afadin is recruited for dephosphorylation by directly binding to the PTPRK D2 pseudophosphatase domain. We mapped this interaction to a putative coiled coil (CC) domain in Afadin that is separated by more than 100 amino acids from the substrate pTyr residue. We identify the residues that define PTP specificity, explaining how Afadin is selectively dephosphorylated by PTPRK yet not by the closely related receptor tyrosine phosphatase PTPRM. Our work demonstrates that PTP substrate specificity can be determined by protein-protein interactions distal to the active site. This explains how PTPRK and other PTPs achieve substrate specificity despite a lack of specific sequence context at the substrate pTyr. Furthermore, by demonstrating that these interactions are phosphorylation-independent and mediated via binding to a non-catalytic domain, we highlight how receptor PTPs could function as intracellular scaffolds in addition to catalyzing protein dephosphorylation.
Assuntos
Proteínas dos Microfilamentos , Proteínas Tirosina Fosfatases , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Especificidade por SubstratoRESUMO
Ir-catalysed borylation of phthalonitrile produces both 4-(Bpin)phthalonitrile (1) and 3,5-bis(Bpin)phthalonitrile (2), which are potential divergent intermediates for the synthesis of functionalized phthalocyanines. To exemplify the utility of 2, we have prepared a series of 3,5-bis-arylphthalonitriles that in turn undergo sterically controlled regioselective cyclotetramization to give previously unknown C4h 1,3,8,10,15,17,22,24-octaarylphthalocyanines.