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1.
Protein Sci ; 33(7): e5075, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38895978

RESUMO

Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.


Assuntos
Piruvato Quinase , Zymomonas , Humanos , Zymomonas/enzimologia , Zymomonas/genética , Zymomonas/química , Zymomonas/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Substituição de Aminoácidos , Estabilidade Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Estabilidade Enzimática , Fígado/enzimologia , Fígado/metabolismo , Fígado/química , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato/química
2.
Cell Death Dis ; 15(6): 408, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862470

RESUMO

The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8-17 °C. In contrast, two non-cancerous mutations, Y103C and Y107C, had only a moderate effect on protein stability. Differential stabilization of the mutants upon treatment with the anticancer agent arsenic trioxide and stibogluconate revealed an interesting proximity effect. Crystallographic studies complemented by MD simulations showed that two of the mutations, Y234C and Y236C, create internal cavities of different size and shape, whereas the others induce unique surface lesions. The mutation-induced pockets in the Y126C and Y205C mutant were, however, relatively small compared with that of the already druggable Y220C mutant. Intriguingly, our structural studies suggest a pronounced plasticity of the mutation-induced pocket in the frequently occurring Y163C mutant, which may be exploited for the development of small-molecule stabilizers. We point out general principles for reactivating thermolabile cancer mutants and highlight special cases where mutant-specific drugs are needed for the pharmacological rescue of p53 function in tumors.


Assuntos
Mutação , Neoplasias , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Humanos , Mutação/genética , Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Trióxido de Arsênio/farmacologia , Simulação de Dinâmica Molecular , Estabilidade Proteica/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química
3.
Protein Sci ; 33(7): e5030, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38864696

RESUMO

Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.


Assuntos
Coagulase , Fibrinogênio , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/química , Coagulase/metabolismo , Coagulase/química , Fibrinogênio/química , Fibrinogênio/metabolismo , Ligação Proteica , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/química , Humanos , Estabilidade Proteica
4.
Protein Sci ; 33(7): e5031, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38864692

RESUMO

Proteins are constantly undergoing folding and unfolding transitions, with rates that determine their homeostasis in vivo and modulate their biological function. The ability to optimize these rates without affecting overall native stability is hence highly desirable for protein engineering and design. The great challenge is, however, that mutations generally affect folding and unfolding rates with inversely complementary fractions of the net free energy change they inflict on the native state. Here we address this challenge by targeting the folding transition state (FTS) of chymotrypsin inhibitor 2 (CI2), a very slow and stable two-state folding protein with an FTS known to be refractory to change by mutation. We first discovered that the CI2's FTS is energetically taxed by the desolvation of several, highly conserved, charges that form a buried salt bridge network in the native structure. Based on these findings, we designed a CI2 variant that bears just four mutations and aims to selectively stabilize the FTS. This variant has >250-fold faster rates in both directions and hence identical native stability, demonstrating the success of our FTS-centric design strategy. With an optimized FTS, CI2 also becomes 250-fold more sensitive to proteolytic degradation by its natural substrate chymotrypsin, and completely loses its activity as inhibitor. These results indicate that CI2 has been selected through evolution to have a very unstable FTS in order to attain the kinetic stability needed to effectively function as protease inhibitor. Moreover, the CI2 case showcases that protein (un)folding rates can critically pivot around a few key residues-interactions, which can strongly modify the general effects of known structural factors such as domain size and fold topology. From a practical standpoint, our results suggest that future efforts should perhaps focus on identifying such critical residues-interactions in proteins as best strategy to significantly improve our ability to predict and engineer protein (un)folding rates.


Assuntos
Mutação , Dobramento de Proteína , Estabilidade Proteica , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Modelos Moleculares , Cinética , Conformação Proteica , Peptídeos
5.
Cell Mol Life Sci ; 81(1): 257, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874784

RESUMO

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.


Assuntos
DNA , Evolução Molecular Direcionada , Edição de Genes , Simulação de Dinâmica Molecular , Mutação , DNA/metabolismo , DNA/genética , DNA/química , Edição de Genes/métodos , Adenina/metabolismo , Adenina/química , Estabilidade Proteica , Ligação Proteica , Eletricidade Estática , Sistemas CRISPR-Cas/genética
6.
Cell Commun Signal ; 22(1): 303, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38831321

RESUMO

BACKGROUND: While previous studies have primarily focused on Glucose transporter type 1 (GLUT1) related glucose metabolism signaling, we aim to discover if GLUT1 promotes tumor progression through a non-metabolic pathway. METHODS: The RNA-seq and microarray data were comprehensively analyzed to evaluate the significance of GLUT1 expression in lung adenocarcinoma (LUAD). The cell proliferation, colony formation, invasion, and migration were used to test GLUT1 's oncogenic function. Co-immunoprecipitation and mass spectrum (MS) were used to uncover potential GLUT1 interacting proteins. RNA-seq, DIA-MS, western blot, and qRT-PCR to probe the change of gene and cell signaling pathways. RESULTS: We found that GLUT1 is highly expressed in LUAD, and higher expression is related to poor patient survival. GLUT1 knockdown caused a decrease in cell proliferation, colony formation, migration, invasion, and induced apoptosis in LUAD cells. Mechanistically, GLUT1 directly interacted with phosphor-epidermal growth factor receptor (p-EGFR) and prevented EGFR protein degradation via ubiquitin-mediated proteolysis. The GLUT1 inhibitor WZB117 can increase the sensitivity of LUAD cells to EGFR-tyrosine kinase inhibitors (TKIs) Gefitinib. CONCLUSIONS: GLUT1 expression is higher in LUAD and plays an oncogenic role in lung cancer progression. Combining GLUT1 inhibitors and EGFR-TKIs could be a potential therapeutic option for LUAD treatment.


Assuntos
Adenocarcinoma de Pulmão , Proliferação de Células , Receptores ErbB , Transportador de Glucose Tipo 1 , Neoplasias Pulmonares , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Humanos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Fosforilação , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Ligação Proteica , Apoptose , Estabilidade Proteica
7.
Protein Sci ; 33(7): e5074, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38888268

RESUMO

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.


Assuntos
Proteínas do Capsídeo , Capsídeo , Dependovirus , Terapia Genética , Vetores Genéticos , Dependovirus/genética , Dependovirus/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Vetores Genéticos/genética , Terapia Genética/métodos , Capsídeo/química , Capsídeo/metabolismo , Espectrometria de Massa com Troca Hidrogênio-Deutério , Estabilidade Proteica , Humanos , Conformação Proteica , Modelos Moleculares
8.
Nat Commun ; 15(1): 4703, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830868

RESUMO

Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Pulmonares , Fator 2 Relacionado a NF-E2 , Peptidilprolil Isomerase , Estabilidade Proteica , Ubiquitinação , Animais , Feminino , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Progressão da Doença , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Camundongos Nus , Fator 2 Relacionado a NF-E2/metabolismo , Proteólise , Peptidilprolil Isomerase/metabolismo
9.
Curr Microbiol ; 81(7): 211, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38839629

RESUMO

This study aimed to obtain reliable high Vip3A production from Bacillus thuringiensis (Bt) by modifying Vip3A to acquire higher thermostability in a suitable host. Bt117 is a great host for Vip3A production due to protein production consistency, low protease activity in culture media, and large amounts of mostly full-length protein, but it produces Vip3A with lower thermostability (Vip3Aa35). The C-terminal region of Bt117 Vip3A was replaced with that of a Vip3A with higher thermostability (Vip3Aa64 from Bt294) to generate the recombinant Bt117-Vip3Aa64-C. Like the parental strain Bt117, this strain expressed mostly full-length protein and exhibited low protease activity and similar protein expression profiles in culture media but retained greater larvicidal activity upon 37 °C storage like Bt294 Vip3Aa64. Importantly, every culture batch of Bt117-Vip3Aa64-C yielded over 200 mg/l Vip3A, which is a notable improvement over the original Vip3Aa64-producing strain Bt294 where 45% of culture batches failed to produce Vip3A at the same level. Successfully, we combined the superior qualities of two Bt strains, Bt294, which produces thermostable Vip3A but at low and inconsistent levels, and Bt117, which produces Vip3A with low thermostability but at consistently high levels. Protein engineering of Vip3A in Bt117 ultimately yielded an improved strain producing a thermostable Vip3A with reliably high protein production.


Assuntos
Bacillus thuringiensis , Proteínas de Bactérias , Engenharia de Proteínas , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Larva/microbiologia , Estabilidade Proteica
10.
Mol Med ; 30(1): 75, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38834947

RESUMO

BACKGROUND: Liver kinase B1 (LKB1) is frequently mutated in lung adenocarcinoma, and its loss contributes to tumor progression. METHODS: To identify LKB1 downstream genes that promote lung adenocarcinoma aggressiveness, we performed bioinformatical analysis using publicly available datasets. RESULTS: Rab3B was upregulated in LKB1-depleted lung adenocarcinoma cells and suppressed by LKB1 overexpression. CREB protein was enriched at the promoter of Rab3B in lung cancer cells. Silencing of CREB abrogated the upregulation of Rab3B upon LKB1 loss. Immunohistochemistry revealed the elevated expression of Rab3B in lung adenocarcinomas relative to adjacent normal tissues. Upregulation of Rab3B was significantly associated with lymph node metastasis, advanced tumor stage, and reduced overall survival in lung adenocarcinoma patients. Knockdown of Rab3B suppressed and overexpression of Rab3B promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells in vitro. In a mouse xenograft model, Rab3B depletion restrained and Rab3B overexpression augmented the growth of lung adenocarcinoma tumors. Mechanistically, Rab3B interacted with DDX6 and enhanced its protein stability. Ectopic expression of DDX6 significantly promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells. DDX6 knockdown phenocopied the effects of Rab3B depletion on lung adenocarcinoma cells. Additionally, DDX6 overexpression partially rescued the aggressive phenotype of Rab3B-depleted lung adenocarcinoma cells. CONCLUSION: LKB1 deficiency promotes Rab3B upregulation via a CREB-dependent manner. Rab3B interacts with and stabilizes DDX6 protein to accelerate lung adenocarcinoma progression. The Rab3B-DDX6 axis may be potential therapeutic target for lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão , RNA Helicases DEAD-box , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Animais , Feminino , Humanos , Masculino , Camundongos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Quinases Proteína-Quinases Ativadas por AMP/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica
11.
Protein Eng Des Sel ; 372024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38836499

RESUMO

Protein developability is requisite for use in therapeutic, diagnostic, or industrial applications. Many developability assays are low throughput, which limits their utility to the later stages of protein discovery and evolution. Recent approaches enable experimental or computational assessment of many more variants, yet the breadth of applicability across protein families and developability metrics is uncertain. Here, three library-scale assays-on-yeast protease, split green fluorescent protein (GFP), and non-specific binding-were evaluated for their ability to predict two key developability outcomes (thermal stability and recombinant expression) for the small protein scaffolds affibody and fibronectin. The assays' predictive capabilities were assessed via both linear correlation and machine learning models trained on the library-scale assay data. The on-yeast protease assay is highly predictive of thermal stability for both scaffolds, and the split-GFP assay is informative of affibody thermal stability and expression. The library-scale data was used to map sequence-developability landscapes for affibody and fibronectin binding paratopes, which guides future design of variants and libraries.


Assuntos
Fibronectinas , Proteínas Recombinantes de Fusão , Fibronectinas/química , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Engenharia de Proteínas/métodos , Biblioteca de Peptídeos , Estabilidade Proteica , Ligação Proteica , Humanos
12.
Anal Chem ; 96(24): 10003-10012, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38853531

RESUMO

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.


Assuntos
Fragmentos Fc das Imunoglobulinas , Espectrometria de Massas , Desdobramento de Proteína , Proteínas Recombinantes de Fusão , Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/química , Espectrometria de Massas/métodos , Interleucina-10/química , Interleucina-10/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Estabilidade Proteica , Humanos , Imunoglobulina G/química
13.
Biol Direct ; 19(1): 46, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38880876

RESUMO

BACKGROUND: There is growing evidence indicating that deubiquitinating enzymes may contribute to tumor progression and can serve as promising therapeutic targets. METHODS: The overexpression of deubiquitinase OTUD6B in lung adenocarcinoma (LUAD) and its adjacent tissues was analyzed by immunohistochemistry and TCGA/GO database. Survival analysis further supported OTUD6B as a potential target for LUAD treatment. We assessed the effect of OTUD6B on LUAD cell growth using cell viability assays and conducted TUNEL staining, migration, and invasion experiments to investigate the impact of OTUD6B on the apoptosis and metastasis of LUAD cells. Additionally, we established a transplanted tumor model in nude mice to validate our findings in vivo. Finally, using IP mass spectrometry and co-IP experiments, we screened and confirmed the influence of RIPK1 as a substrate of OTUD6B in LUAD. RESULTS: OTUD6B is highly overexpressed in human LUAD and predicts poor prognosis in LUAD patients. OTUD6B knockdown inhibited the proliferation of LUAD cells and enhanced apoptosis and inhibited metastasis in LUAD cells suppressed. A549 xenografts revealed that OTUD6B deletion can slow down tumour growth. Additionally, OTUD6B can bind to RIPK1, reduce its ubiquitination level and increase its protein stability. CONCLUSIONS: Our results suggest that OTUD6B is a promising clinical target for LUAD treatment and that targeting OTUD6B may constitute an effective anti-LUAD strategy.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Camundongos Nus , Proteína Serina-Treonina Quinases de Interação com Receptores , Humanos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Progressão da Doença , Proliferação de Células , Apoptose , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Células A549 , Ubiquitinação , Estabilidade Proteica , Endopeptidases/metabolismo , Endopeptidases/genética
14.
J Cell Biol ; 223(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38874443

RESUMO

N-degrons are short sequences located at protein N-terminus that mediate the interaction of E3 ligases (E3s) with substrates to promote their proteolysis. It is well established that N-degrons can be exposed following protease cleavage to allow recognition by E3s. However, our knowledge regarding how proteases and E3s cooperate in protein quality control mechanisms remains minimal. Using a systematic approach to monitor the protein stability of an N-terminome library, we found that proline residue at the third N-terminal position (hereafter "P+3") promotes instability. Genetic perturbations identified the dipeptidyl peptidases DPP8 and DPP9 and the primary E3s of N-degron pathways, UBR proteins, as regulators of P+3 bearing substrate turnover. Interestingly, P+3 UBR substrates are significantly enriched for secretory proteins. We found that secretory proteins relying on a signal peptide (SP) for their targeting contain a "built-in" N-degron within their SP. This degron becomes exposed by DPP8/9 upon translocation failure to the designated compartments, thus enabling clearance of mislocalized proteins by UBRs to maintain proteostasis.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases , Estabilidade Proteica , Ubiquitina-Proteína Ligases , Humanos , Degrons , Dipeptidases/metabolismo , Dipeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Células HEK293 , Sinais Direcionadores de Proteínas , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética
15.
Biochemistry (Mosc) ; 89(5): 912-922, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38880651

RESUMO

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a binding partner of the phosphatase CD45, but its function remains poorly understood. Its close interaction with CD45 suggests that LPAP may potentially regulate CD45, but direct biochemical evidence for this has not yet been obtained. We found that in the Jurkat lymphoid cells the levels of LPAP and CD45 proteins are interrelated and well correlated with each other. Knockout of LPAP leads to the decrease in the surface expression of CD45, while its overexpression, on the contrary, caused its increase. No such correlation was found in the non-lymphoid K562 cells. We hypothesize that LPAP regulates expression level of CD45 and thus can affect lymphocyte activation.


Assuntos
Antígenos Comuns de Leucócito , Humanos , Antígenos Comuns de Leucócito/metabolismo , Células Jurkat , Células K562 , Estabilidade Proteica , Fosfoproteínas/metabolismo , Fosfoproteínas/genética
16.
Cell Mol Life Sci ; 81(1): 251, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38847937

RESUMO

The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.


Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Instabilidade Genômica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Domínios Proteicos , Estabilidade Proteica , Mutação , Linhagem Celular , DNA Helicases
17.
Acta Pharm ; 74(2): 289-300, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38815206

RESUMO

At present, society has embraced the fact apropos population aging and climate changes, that demand, amongst others, innovative pharmaceutical technologies, emphasising the development of patient-specific delivery systems and thus the provision of efficient and sustainable drugs. Protein drugs for subcutaneous administration, by allowing less frequent application, represent one of the most important parts of the pharmaceutical field, but their development is inevitably faced with obstacles in providing protein stability and suitable formulation viscosity. To gain further knowledge and fill the gaps in the already constructed data platform for the development of monoclonal antibody formulations, we designed a study that examines small model proteins, i.e., bovine serum albumin. The main aim of the presented work is to evaluate the effect of protein concentrations on critical quality attributes of both, pre-lyophilised liquid formulations, and lyophilised products. Through the study, the hypothesis that increasing protein concentration leads to higher viscosity and higher reconstitution time without affecting the stability of the protein was confirmed. The most important finding is that sucrose plays a key role in the lyophilisation of investigated protein, nevertheless, it can be predicted that, to ensure the beneficial effect of mannitol, its amount has to prevail over the amount of sucrose.


Assuntos
Composição de Medicamentos , Liofilização , Soroalbumina Bovina , Soroalbumina Bovina/química , Viscosidade , Composição de Medicamentos/métodos , Humanos , Sacarose/química , Estabilidade de Medicamentos , Química Farmacêutica/métodos , Excipientes/química , Manitol/química , Estabilidade Proteica
18.
Biochem Biophys Res Commun ; 721: 150109, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-38762932

RESUMO

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.


Assuntos
Cálcio , Endopeptidase K , Estabilidade Enzimática , Simulação de Dinâmica Molecular , Estabilidade Proteica , Endopeptidase K/metabolismo , Endopeptidase K/química , Cálcio/metabolismo , Cálcio/química , Desenho Assistido por Computador , Mutação , Sítios de Ligação , Engenharia de Proteínas/métodos , Conformação Proteica
19.
Food Res Int ; 186: 114337, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38729718

RESUMO

A major concern for wineries is haze formation in white wines due to protein instability. Despite its prevalent use, the conventional bentonite method has shortcomings, including potential alteration of color and aroma, slow processing times, and notable wine wastage. Zirconium oxide (ZrO2) effectively removes proteins without affecting wine characteristics. However, producing cost-effective ZrO2 materials with efficient protein removal capabilities poses a significant challenge. This research aims to assess the viability of designing a porous material impregnated with zirconia to remove turbidity-causing proteins effectively. For this purpose, the support material alone (Al2O3) and the zirconia-impregnated support (ZrO2/Al2O3) were subjected to different calcination temperatures. It was observed that high-temperature treatments (750 °C) enhanced wine stability and protein adsorption capacity. The optimal adsorbent achieved a notable reduction in turbidity, decreasing the ΔNTU from 42 to 18, alongside a significant 44 % reduction in the total protein content, particularly affecting proteins in the molecular weight range of 10 to 70 kDa. This result is attributed to modifying the textural properties of ZrO2/Al2O3, characterized by the reduction of acidic sites, augmented pore diameters from 4.81 to 7.74 nm, and the emergence of zirconia clusters across the surface of the porous support. In summary, this study presents the first application of zirconia on the alumina support surface for protein stabilization in white wine. Combining ZrO2/Al2O3 and a high-temperature treatment emerges as a promising, cost-efficient, and environmentally sustainable strategy for protein removal in white wine.


Assuntos
Óxido de Alumínio , Vinho , Zircônio , Vinho/análise , Zircônio/química , Óxido de Alumínio/química , Adsorção , Estabilidade Proteica , Temperatura Alta , Manipulação de Alimentos/métodos
20.
Aging (Albany NY) ; 16(10): 8965-8979, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38787373

RESUMO

BACKGROUND: Bone formation and homeostasis are greatly dependent on the osteogenic differentiation of human bone marrow stem cells (BMSCs). Therefore, revealing the mechanisms underlying osteogenic differentiation of BMSCs will provide new candidate therapeutic targets for osteoporosis. METHODS: The osteogenic differentiation of BMSCs was measured by analyzing ALP activity and expression levels of osteogenic markers. Cellular Fe and ROS levels and cell viability were applied to evaluate the ferroptosis of BMSCs. qRT-PCR, Western blotting, and co-immunoprecipitation assays were harnessed to study the molecular mechanism. RESULTS: The mRNA level of CRYAB was decreased in the plasma of osteoporosis patients. Overexpression of CRYAB increased the expression of osteogenic markers including OCN, OPN, RUNX2, and COLI, and also augmented the ALP activity in BMSCs, on the contrary, knockdown of CRYAB had opposite effects. IP-MS technology identified CRYAB-interacted proteins and further found that CRYAB interacted with ferritin heavy chain 1 (FTH1) and maintained the stability of FTH1 via the proteasome mechanism. Mechanically, we unraveled that CRYAB regulated FTH1 protein stability in a lactylation-dependent manner. Knockdown of FTH1 suppressed the osteogenic differentiation of BMSCs, and increased the cellular Fe and ROS levels, and eventually promoted ferroptosis. Rescue experiments revealed that CRYAB suppressed ferroptosis and promoted osteogenic differentiation of BMSCs via regulating FTH1. The mRNA level of FTH1 was decreased in the plasma of osteoporosis patients. CONCLUSIONS: Downregulation of CRYAB boosted FTH1 degradation and increased cellular Fe and ROS levels, and finally improved the ferroptosis and lessened the osteogenic differentiation of BMSCs.


Assuntos
Diferenciação Celular , Ferroptose , Osteogênese , Osteoporose , Humanos , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Osteoporose/patologia , Células-Tronco Mesenquimais/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/genética , Ferritinas/metabolismo , Estabilidade Proteica , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Células da Medula Óssea/metabolismo , Feminino , Oxirredutases
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