RESUMEN
To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.
Asunto(s)
Proteínas Bacterianas , Chlamydophila pneumoniae , Endocitosis , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42 , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteína de Unión al GTP cdc42/metabolismo , Membrana Celular/metabolismo , Chlamydophila pneumoniae/metabolismo , Cristalografía por Rayos X , Células HeLa , Interacciones Huésped-Patógeno , Imitación Molecular , Unión Proteica , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/química , Nexinas de Clasificación/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Animales , RatasRESUMEN
Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Síndrome de Noonan , Proteínas ras , Humanos , Síndrome de Noonan/genética , Síndrome de Noonan/patología , Síndrome de Noonan/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Proteínas ras/metabolismo , Proteínas ras/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Mutación/genética , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Cardiomiopatía Hipertrófica/metabolismo , Polimerizacion , Sistemas CRISPR-Cas/genética , Proteolisis , Mutación Missense , Multimerización de Proteína , Genes Recesivos , FenotipoRESUMEN
Small GTPases switch between GDP- and GTP-bound states during cell signaling. The ADP-ribosylation factor (ARF) family of small GTPases is involved in vesicle trafficking. Although evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5 (TTN5; also known as HALLIMASCH, ARL2 and ARLC1) from Arabidopsis thaliana, and two TTN5 proteins with point mutants in conserved residues, TTN5T30N and TTN5Q70L, that were expected to be unable to perform nucleotide exchange and GTP hydrolysis, respectively. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 by in situ immunolocalization in Arabidopsis seedlings and through use of a transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and that it dynamically associates with membranes of vesicles, Golgi stacks and multivesicular bodies. Although TTN5Q70L mirrored wild-type TTN5 behavior, the TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with its membrane dynamics, and TTN5 likely has a role in vesicle transport within the endomembrane system.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Guanosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Hidrólisis , Aparato de Golgi/metabolismoRESUMEN
Small GTPases comprise key proteins in signal transduction that function by conformational switching ability between GDP- and GTP-bound states. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking and cellular functions. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized functional properties and cellular localization of the essential small ARF-like GTPase TITAN5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. TTN5 showed rapid guanine nucleotide exchange capacity comparable to that of human counterparts, but a remarkably low GTP hydrolysis reaction. A TTN5Q70L mutant had enhanced nucleotide exchange activity, indicative of intracellular activation, while TTN5T30N with fast nucleotide dissociation can be considered a dominant-negative form. This suggests that TTN5 is present in GTP-loaded active form in the cells. YFP-tagged TTN5 and the two derived mutant variants were located at multiple sites of the endomembrane system in the epidermis of Arabidopsis seedlings and Nicotiana benthamiana leaves. While YFP-TTN5 and YFP-TTN5Q70L were highly mobile in the cells, mobility was reduced for TTN5T30N. Colocalization with endomembrane markers in combination with pharmacological treatments resolved localization at membrane sites and showed that YFP-TTN5 and YFP-TTN5Q70L were located in Golgi stacks, multivesicular bodies, while this was less the case for YFP-TTN5T30N. On the other hand, all three TTN5 forms were located at the plasma membrane. Hence, the unusual capacity of rapid nucleotide exchange activity of the small ARF-like GTPase TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system.
RESUMEN
Herein, we designed a DNA framework-based intelligent nanorobot using toehold-mediated strand displacement reaction-based molecular programming and logic gate operation for the selective and synchronous detection of miR21 and miR125b, which are known as significant cancer biomarkers. Moreover, to investigate the applicability of our design, DNA nanorobots were implemented as capping agents onto the pores of MSNs. These agents can develop a logic-responsive hybrid nanostructure capable of specific drug release in the presence of both targets. The prosperous synthesis steps were verified by FTIR, XRD, BET, UV-visible, FESEM-EDX mapping, and HRTEM analyses. Finally, the proper release of the drug in the presence of both target microRNAs was studied. This Hybrid DNA Nanostructure was designed with the possibility to respond to any target oligonucleotides with 22 nucleotides length.
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MicroARNs , Nanoestructuras , Neoplasias , Humanos , MicroARNs/análisis , MicroARNs/genética , Biomarcadores de Tumor/genética , Neoplasias/genética , ADN/química , Nanoestructuras/químicaRESUMEN
KRAS is one of the most widely prevalent proto-oncogenes in human cancers. The constitutively active KRAS oncoprotein contributes to both tumor onset and cancer development by promoting cell proliferation and anchorage-independent growth in a MAPK pathway-dependent manner. The expression of microRNAs (miRNAs) and the KRAS oncogene are known to be dysregulated in various cancers, while long noncoding RNAs (lncRNAs) can act as regulators of the miRNAs targeting KRAS oncogene in different cancers and have gradually become a focus of research in recent years. In this review article, we summarize recent advances in the research on lncRNAs that have sponging effects on KRAS-targeting miRNAs as crucial mediators of KRAS expression in different cell types and organs. A deeper understanding of lncRNA function in KRAS-driven cancers is of major fundamental importance and will provide a valuable clinical tool for the diagnosis, prognosis, and eventual treatment of cancers.
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MicroARNs , Neoplasias , ARN Largo no Codificante , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
RHO GTPases are critical signal transducers that regulate cell adhesion, polarity, and migration through multiple signaling pathways. While all these cellular processes are crucial for the maintenance of normal cell homeostasis, disturbances in RHO GTPase-associated signaling pathways contribute to different human diseases, including many malignancies. Several members of the RHO GTPase family are frequently upregulated in human tumors. Abnormal gene regulation confirms the pivotal role of lncRNAs as critical gene regulators, and thus, they could potentially act as oncogenes or tumor suppressors. lncRNAs most likely act as sponges for miRNAs, which are known to be dysregulated in various cancers. In this regard, the significant role of miRNAs targeting RHO GTPases supports the view that the aberrant expression of lncRNAs may reciprocally change the intensity of RHO GTPase-associated signaling pathways. In this review article, we summarize recent advances in lncRNA research, with a specific focus on their sponge effects on RHO GTPase-targeting miRNAs to crucially mediate gene expression in different cancer cell types and tissues. We will focus in particular on five members of the RHO GTPase family, including RHOA, RHOB, RHOC, RAC1, and CDC42, to illustrate the role of lncRNAs in cancer progression. A deeper understanding of the widespread dysregulation of lncRNAs is of fundamental importance for confirmation of their contribution to RHO GTPase-dependent carcinogenesis.