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1.
Cell Mol Life Sci ; 81(1): 139, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38480559

RESUMO

Neurotoxic amyloid-ß (Aß) peptides cause neurodegeneration in Alzheimer's disease (AD) patients' brains. They are released upon proteolytic processing of the amyloid precursor protein (APP) extracellularly at the ß-secretase site and intramembranously at the γ-secretase site. Several AD mouse models were developed to conduct respective research in vivo. Most of these classical models overexpress human APP with mutations driving AD-associated pathogenic APP processing. However, the resulting pattern of Aß species in the mouse brains differs from those observed in AD patients' brains. Particularly mutations proximal to the ß-secretase cleavage site (e.g., the so-called Swedish APP (APPswe) fostering Aß1-x formation) lead to artificial Aß production, as N-terminally truncated Aß peptides are hardly present in these mouse brains. Meprin ß is an alternative ß-secretase upregulated in brains of AD patients and capable of generating N-terminally truncated Aß2-x peptides. Therefore, we aimed to generate a mouse model for the production of so far underestimated Aß2-x peptides by conditionally overexpressing meprin ß in astrocytes. We chose astrocytes as meprin ß was detected in this cell type in close proximity to Aß plaques in AD patients' brains. The meprin ß-overexpressing mice showed elevated amyloidogenic APP processing detected with a newly generated neo-epitope-specific antibody. Furthermore, we observed elevated Aß production from endogenous APP as well as AD-related behavior changes (hyperlocomotion and deficits in spatial memory). The novel mouse model as well as the established tools and methods will be helpful to further characterize APP cleavage and the impact of different Aß species in future studies.


Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Camundongos , Animais , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Astrócitos/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Proteólise , Encéfalo/metabolismo
2.
Int J Mol Sci ; 25(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38473819

RESUMO

Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein-protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes.


Assuntos
Relógios Circadianos , Neurospora crassa , Proteólise , Processamento de Proteína Pós-Traducional , Fosforilação , Proteínas CLOCK , Ritmo Circadiano , Proteínas Fúngicas
3.
Int J Mol Sci ; 25(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474064

RESUMO

Our previous study has reported that metastasis-associated protein 2 (MTA2) plays essential roles in tumorigenesis and aggressiveness of gastric cancer (GC). However, the underlying molecular mechanisms of MTA2-mediated GC and its upstream regulation mechanism remain elusive. In this study, we identified a novel circular RNA (circRNA) generated from the MTA2 gene (circMTA2) as a crucial regulator in GC progression. CircMTA2 was highly expressed in GC tissues and cell lines, and circMTA2 promoted the proliferation, invasion, and metastasis of GC cells both in vitro and in vivo. Mechanistically, circMTA2 interacted with ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) to restrain MTA2 ubiquitination and stabilize MTA2 protein expression, thereby facilitating tumor progression. Moreover, circMTA2 was mainly encapsulated and transported by exosomes to promote GC cell progression. Taken together, these findings uncover that circMTA2 suppresses MTA2 degradation by interacting with UCHL3, thereby promoting GC progression. In conclusion, we identified a cancer-promoting axis (circMTA2/UCHL3/MTA2) in GC progression, which paves the way for us to design and synthesize targeted inhibitors as well as combination therapies.


Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patologia , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Histona Desacetilases/metabolismo , Proteólise , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Ubiquitina Tiolesterase/metabolismo
4.
Nat Commun ; 15(1): 2005, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443338

RESUMO

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.


Assuntos
Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteólise , Dimerização , Descoberta de Drogas
5.
PLoS Biol ; 22(3): e3002548, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38452120

RESUMO

Proteasomes and autophagy constitute the 2 main proteolytic machineries for cytoplasmic content. A new study in PLOS Biology now demonstrates that autophagy stimulation alters proteasome composition, degrading hyperactive immunoproteasomes and thereby limiting inflammation.


Assuntos
Inflamação , Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Macrófagos/metabolismo , Autofagia
6.
Nat Commun ; 15(1): 2064, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38453899

RESUMO

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.


Assuntos
Serina Endopeptidases , Serina Proteases , Dimerização , Serina Endopeptidases/metabolismo , Proteólise , Replicação do DNA , Serina
7.
Science ; 383(6688): eadk4422, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38484051

RESUMO

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.


Assuntos
Evolução Molecular Direcionada , Proteólise , Ubiquitina-Proteína Ligases , Dedos de Zinco , Microscopia Crioeletrônica , Talidomida/química , Ubiquitina-Proteína Ligases/química , Ubiquitinação , Dedos de Zinco/genética , Quimera de Direcionamento de Proteólise , Evolução Molecular Direcionada/métodos , Humanos
8.
Bioorg Med Chem Lett ; 102: 129680, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38428537

RESUMO

Dihydroquinolizinones (DHQs) that inhibit cellular polyadenylating polymerases 5 and 7 (PAPD5 & 7), such as RG7834, have been shown to inhibit both hepatitis A (HAV) and hepatitis B virus (HBV) in vitro and in vivo. In this report, we describe RG7834-based proteolysis-targeting chimeras (PROTACs), such as compound 12b, (6S)-9-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-21-oxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan-25-yl)oxy)-6-isopropyl-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinoline-3-carboxylic acid. The PROTAC DHQs described here inhibited an HAV reporter virus in vitro with an IC50 of 277 nM. Although the PROTAC DHQs were also inhibitory to HBV, their activities were substantially less potent against HBV in vitro, being in the 10 to 20 µM range, based on the reduction of HBsAg and HBV mRNA levels. Importantly, unlike RG7834, the incubation of cells in vitro with PROTAC DHQ 12b resulted in the degradation of PAPD5, as expected for a PROTAC compound, but curiously not PAPD7. PAPD5 polypeptide degradation was prevented when a proteasome inhibitor, epoxomicin, was used, indicating that proteasome mediated proteolysis was associated with the observed activities of 12b. Taken together, these data show that 12b is the first example of a PROTAC that suppresses both HAV and HBV that is based on a small molecule warhead. The possibility that it has mechanisms that differ from its parent compound, RG7834, and has clinical value, is discussed.


Assuntos
Vírus da Hepatite A , Vírus da Hepatite B , Proteólise , Complexo de Endopeptidases do Proteassoma
9.
Food Res Int ; 182: 114146, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38519176

RESUMO

This study evaluated the nutritional quality of different microbial biomass samples by assessing their protein digestibility and carbohydrate fermentability in the colon using in vitro methods. Four microbial samples were produced: one hydrogen-oxidizing bacterial strain (Nocardioides nitrophenolicus KGS-27), two strains of filamentous fungi (Rhizopus oligosporus and Paecilomyces variotii), and one yeast strain (Rhodotorula babjevae). The microorganisms were grown in bioreactors, harvested and dried before analysis. The commercial fungal product Quorn was used as a reference. The protein digestibility of the microbial samples was analysed using the INFOGEST in vitro model, followed by quantification of N-terminal amine groups. An in vitro faecal fermentation experiment was also performed to evaluate the degradation of carbohydrates in microbial biomass samples and formation of short-chain fatty acids (SCFA). The fungal biomass samples had higher protein hydrolysis (60-75 %) than the bacterial sample (12 %) and Quorn (45 %), while the yeast biomass had the highest protein digestibility (85 %). Heat-treatment of the biomass significantly reduced its protein digestibility. Total dietary fibre (DF) content of fungal biomass was 31 - 43 %(DW), mostly insoluble, whereas the bacterial biomass contained mainly soluble DF (total DF: 25.7 %, of which 23.5 % were soluble and 2.2 % insoluble). After 24 h of colonic in vitro fermentation, SCFA production from the biomass of Paecilomyces, Quorn and Rhodotorula was similar to that of wheat bran, while 17 % and 32 % less SCFA were produced from the biomass of Rhizopus and the bacterial strain, respectively. Further studies are needed to clarify the reasons for the observed differences in protein digestibility and DF fermentability, especially regarding the cell wall structures and role of post-processing.


Assuntos
Fibras na Dieta , Ácidos Graxos Voláteis , Fermentação , Proteólise , Biomassa , Fibras na Dieta/análise , Ácidos Graxos Voláteis/metabolismo , Bactérias/metabolismo , Colo/metabolismo , Fungos/metabolismo
10.
J Hematol Oncol ; 17(1): 14, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38520028

RESUMO

Protein degraders, emerging as a novel class of therapeutic agents, have gained widespread attention due to their advantages. They have several advantages over traditional small molecule inhibitors, including high target selectivity and ability to target "undruggable" targets and overcome inhibitor drug resistance. Tremendous research and development efforts and massive investment have resulted in rapid advancement of protein degrader drug discovery in recent years. Here, we overview the latest clinical and preclinical updates on protein degraders presented at the 2023 ASH Annual Meeting.


Assuntos
Neoplasias Hematológicas , Humanos , Proteólise , Neoplasias Hematológicas/tratamento farmacológico , Descoberta de Drogas
12.
Plant Cell Rep ; 43(4): 105, 2024 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-38522062

RESUMO

KEY MESSAGE: A recently reported Pijx gene interacts and promotes the ATPb degradation through 26 proteasomal pathways activate OsRbohC mediated ROS burst, leading to broad-spectrum rice blast resistance in seedling and panicle.


Assuntos
Plântula , Plântula/genética , Proteólise
13.
Biochem Biophys Res Commun ; 706: 149730, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38461648

RESUMO

Peptides linking well-folded and non-interacting domains in fusion proteins can undergo proteolytic degradation. This leads to physical separation of the domains that were originally sought to be joined. In order to identify characteristics that determine linker degradation propensity, we selected a pair of thermostable, proteolytically-resistant domains, and joined them using five different linkers. We then assessed linker degradation propensities through size-exclusion chromatography, and denaturing and non-denaturing electrophoresis. The domains used were Coh2, an all-beta cohesin from C. thermocellum CipA, and BSX, a beta/alpha barrel xylanase from Bacillus sp. NG-27, while the linkers used were Rigid (3 repeats of N-EAAAK-C), Flexible (two repeats of N-SGGGG-C), Nat-full (42 residues of a Coh2-adjacent linker from CipA), Nat-half (a 21 residues-long derivative of Nat-full) and Nat-quarter (a 9 residues-long derivative of Nat-full). Both with proteolysis effected by proteases present in the environment, and with an exogenously-added protease (Subtilisin A), we found that Flexible underwent little or no degradation, whereas linkers of comparable length like Nat-quarter or Rigid underwent extensive degradation, as did longer linkers like Nat-Half and Nat-Full. Our analyses disfavor the likelihood of the sequence of Flexible being naturally resistant to proteolysis, and instead favor the explanation that the flexibility of Flexible facilitates movements of Coh2 relative to BSX which then serve to sterically prevent the approach of proteases. Thus, the construct incorporating Flexible appears to behave like a 'nunchuck' in which rods/spheres flanking a chain collide with approaching swords that are capable of severing the chain, to prevent severance.


Assuntos
Peptídeo Hidrolases , Peptídeos , Propionatos , Proteólise , Peptídeos/química , Indóis , Endopeptidases
14.
Biochem Biophys Res Commun ; 706: 149746, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38461646

RESUMO

Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles ß-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.


Assuntos
Peptídeo Hidrolases , Peptídeos , Peptídeos/química , Peptídeo Hidrolases/metabolismo , Endopeptidases/metabolismo , Proteólise
15.
Proc Natl Acad Sci U S A ; 121(13): e2320053121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38513100

RESUMO

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.


Assuntos
Lisossomos , Humanos , Células HEK293 , Proteólise
16.
BMC Musculoskelet Disord ; 25(1): 227, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38509535

RESUMO

BACKGROUND: Osteoarthritis (OA) represents a prominent etiology of considerable pain and disability, and conventional imaging methods lack sensitivity in diagnosing certain types of OA. Therefore, there is a need to identify highly sensitive and efficient biomarkers for OA diagnosis. Zinc ions feature in the pathogenesis of OA. This work aimed to investugate the role of zinc metabolism-related genes (ZMRGs) in OA and the diagnostic characteristics of key genes. METHODS: We obtained datasets GSE169077 and GSE55235 from the Gene Expression Omnibus (GEO) and obtained ZMRGs from MSigDB. Differential expression analysis was conducted on the GSE169077 dataset using the limma R package to identify differentially expressed genes (DEGs), and the intersection of DEGs and ZMRGs yielded zinc metabolism differential expression-related genes (ZMRGs-DEGs). The clusterProfiler R package was employed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of ZMRGs-DEGs. Potential small molecule drugs were predicted using the CMap database, and immune cell infiltration and function in OA individuals were analyzed using the ssGSEA method. Protein-protein interaction (PPI) networks were constructed to detect Hub genes among ZMRGs-DEGs. Hub gene expression levels were analyzed in the GSE169077 and GSE55235 datasets, and their diagnostic characteristics were assessed using receiver operating characteristic (ROC) curves. The gene-miRNA interaction network of Hub genes was explored using the gene-miRNA interaction network website. RESULTS: We identified 842 DEGs in the GSE169077 dataset, and their intersection with ZMRGs resulted in 46 ZMRGs-DEGs. ZMRGs-DEGs were primarily enriched in functions such as collagen catabolic processes, extracellular matrix organization, metallopeptidase activity, and pathways like the IL-17 signaling pathway, Nitrogen metabolism, and Relaxin signaling pathway. Ten potential small-molecule drugs were predicted using the CMap database. OA patients exhibited distinct immune cell abundance and function compared to healthy individuals. We identified 4 Hub genes (MMP2, MMP3, MMP9, MMP13) through the PPI network, which were highly expressed in OA and demonstrated good diagnostic performance. Furthermore, two closely related miRNAs for each of the 4 Hub genes were identified. CONCLUSION: 4 Hub genes were identified as potential diagnostic biomarkers and therapeutic targets for OA.


Assuntos
MicroRNAs , Zinco , Humanos , Proteólise , Bases de Dados Factuais , Biomarcadores , Biologia Computacional , Perfilação da Expressão Gênica
17.
J Am Chem Soc ; 146(11): 7584-7593, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38469801

RESUMO

Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.


Assuntos
DNA Forma Z , Quimera de Direcionamento de Proteólise , Proteólise , Adenosina Desaminase/metabolismo , RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo
18.
Biochem Soc Trans ; 52(1): 431-440, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38329179

RESUMO

The amount of the low-density lipoprotein receptor (LDLR) on the surface of hepatocytes is the primary determinant of plasma low-density lipoprotein (LDL)-cholesterol level. Although the synthesis and cellular trafficking of the LDLR have been well-documented, there is growing evidence of additional post-translational mechanisms that regulate or fine tune the surface availability of the LDLR, thus modulating its ability to bind and internalise LDL-cholesterol. Proprotein convertase subtilisin/kexin type 9 and the asialoglycoprotein receptor 1 both independently interact with the LDLR and direct it towards the lysosome for degradation. While ubiquitination by the E3 ligase inducible degrader of the LDLR also targets the receptor for lysosomal degradation, ubiquitination of the LDLR by a different E3 ligase, RNF130, redistributes the receptor away from the plasma membrane. The activity of the LDLR is also regulated by proteolysis. Proteolytic cleavage of the transmembrane region of the LDLR by γ-secretase destabilises the receptor, directing it to the lysosome for degradation. Shedding of the extracellular domain of the receptor by membrane-type 1 matrix metalloprotease and cleavage of the receptor in its LDL-binding domain by bone morphogenetic protein-1 reduces the ability of the LDLR to bind and internalise LDL-cholesterol at the cell surface. A better understanding of how the activity of the LDLR is regulated will not only unravel the complex biological mechanisms controlling LDL-cholesterol metabolism but also could help inform the development of alternative pharmacological intervention strategies for the treatment of hypercholesterolaemia.


Assuntos
Colesterol , Receptores de LDL , Receptores de LDL/metabolismo , LDL-Colesterol , Proteólise , Hepatócitos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
19.
Nat Commun ; 15(1): 1531, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378719

RESUMO

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.


Assuntos
Colágeno , Matriz Extracelular , Animais , Humanos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Proteólise , Pulmão/patologia , Mamíferos/metabolismo , Proteínas/metabolismo
20.
Genome Biol ; 25(1): 58, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409044

RESUMO

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.


Assuntos
Ácidos Indolacéticos , Humanos , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/metabolismo , Proteínas/metabolismo , Linhagem Celular , Proteólise
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