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1.
Nat Commun ; 15(1): 1007, 2024 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-38307855

RESUMEN

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.


Asunto(s)
Actinas , Proteínas del Ojo , Reguladores de Proteínas de Unión al GTP , Fosfoproteínas , Pliegue de Proteína , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Chaperoninas/metabolismo , Adenosina Trifosfato/metabolismo , Chaperonina con TCP-1/metabolismo
2.
Mol Cell ; 83(17): 3123-3139.e8, 2023 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-37625406

RESUMEN

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.


Asunto(s)
Chaperonina con TCP-1 , Actinas , Chaperonina con TCP-1/química , Chaperonina con TCP-1/metabolismo , Humanos , Animales
3.
Nat Commun ; 13(1): 1226, 2022 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-35264557

RESUMEN

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A. A cryo-EM structure of PA26E102A-h20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.


Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteínas , Citoplasma/metabolismo , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Relación Estructura-Actividad
4.
Nat Chem Biol ; 15(8): 786-794, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31320752

RESUMEN

Protein-protein interactions between E3 ubiquitin ligases and protein termini help shape the proteome. These interactions are sensitive to proteolysis, which alters the ensemble of cellular N and C termini. Here we describe a mechanism wherein caspase activity reveals latent C termini that are then recognized by the E3 ubiquitin ligase CHIP. Using expanded knowledge of CHIP's binding specificity, we predicted hundreds of putative interactions arising from caspase activity. Subsequent validation experiments confirmed that CHIP binds the latent C termini at tauD421 and caspase-6D179. CHIP binding to tauD421, but not tauFL, promoted its ubiquitination, while binding to caspase-6D179 mediated ubiquitin-independent inhibition. Given that caspase activity generates tauD421 in Alzheimer's disease (AD), these results suggested a concise model for CHIP regulation of tau homeostasis. Indeed, we find that loss of CHIP expression in AD coincides with the accumulation of tauD421 and caspase-6D179. These results illustrate an unanticipated link between caspases and protein homeostasis.


Asunto(s)
Caspasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Caspasas/genética , Línea Celular Tumoral , Cristalografía por Rayos X , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Humanos , Unión Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinación
5.
Transl Res ; 198: 48-57, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30244692

RESUMEN

Neurodegenerative diseases are a class of age-associated proteopathies characterized by the accumulation of misfolded and/or aggregation-prone proteins. This imbalance has been attributed, in part, to an age-dependent decay in the capacity of protein turnover. Most proteins are degraded by the ubiquitin-proteasome system (UPS), which is composed of ubiquitin ligases and regulatory particles, such as the 19S, that deliver cargo to the proteolytically active 20S proteasome (20S) core. However, a subset of clients, especially intrinsically disordered proteins (IDPs), are also removed by the action of the ubiquitin-independent proteasome system (UIPS). What are the specific contributions of the UPS and UIPS in the context of neurodegeneration? Here, we explore how age-associated changes in the relative contribution of the UPS and UIPS, combined with the IDP-like structure of many neurodegenerative disease-associated proteins, might contribute. Strikingly, the 20S has been shown to predominate in older neurons and to preferentially act on relevant substrates, such as synuclein and tau. Moreover, pharmacological activation of the 20S has been shown to accelerate removal of aggregation-prone proteins in some models. Together, these recent studies are turning attention to the 20S and the UIPS as potential therapeutic targets in neurodegeneration.


Asunto(s)
Activadores de Enzimas/uso terapéutico , Terapia Molecular Dirigida , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Envejecimiento/metabolismo , Animales , Activadores de Enzimas/farmacología , Humanos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Especificidad por Sustrato
6.
Chem Biol ; 20(7): 888-902, 2013 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-23890007

RESUMEN

PUMA is a proapoptotic BCL-2 family member that drives the apoptotic response to a diversity of cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent proapoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally stabilized PUMA BH3 helices that, in addition to broadly targeting antiapoptotic proteins, directly bind to proapoptotic BAX. NMR, photocrosslinking, and biochemical analyses revealed that PUMA SAHBs engage an α1/α6 trigger site on BAX to initiate its functional activation. We further demonstrated that a cell-permeable PUMA SAHB analog induces apoptosis in neuroblastoma cells and, like expressed PUMA protein, engages BCL-2, MCL-1, and BAX. Thus, we find that PUMA BH3 is a dual antiapoptotic inhibitor and proapoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Activación Enzimática , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Porosidad , Estructura Terciaria de Proteína , Proteómica , Especificidad por Sustrato , Proteína X Asociada a bcl-2/metabolismo
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