Death associated protein like 1 acts as a novel tumor suppressor in melanoma by increasing the stability of P21 protein.

Melanoma is a primary malignant tumor with high lethality, which occurs in the skin and eye tissues, while the molecular mechanisms of melanomagenesis remain largely unknown. Here, we show that death-associated protein-like 1 (DAPL1) expression is lower in melanoma tissues than in paracancerous tissues or nevus tissues, and Uveal melanoma patients with lower DAPL1 expression have a poorer survival rate than those with higher expression of DAPL1. Overexpression of DAPL1 inhibits proliferation of cultured melanoma cells, whereas knockdown of DAPL1 increases cell proliferation. Tumor transplantation experiment results also demonstrate that DAPL1 inhibits tumorigenesis of melanoma cells both in subretinal and subcutaneous tissues of nude mice in vivo. Finally, DAPL1 inhibits proliferation of melanoma cells by increasing the protein level of P21 via decreasing the ubiquitin mediated degradation of P21 and promoting its stability. Conversely, knockdown of P21 neutralizes the effects of inhibition of DAPL1 on melanoma cell proliferation and enhances the severity of melanoma tumorigenesis. These results suggest that DAPL1 is a novel melanoma tumor suppressor gene and thus a potential therapeutic target for melanoma.

LncRNA Snhg12/IGFBP3 axis is involved in liver fibrosis by promoting the proliferation and activation of mouse hepatic stellate cells.

Liver fibrosis is a persistent damage repair response triggered by various injury factors, which leads to an abnormal accumulation of extracellular matrix within liver tissue samples. The current clinical treatment of liver fibrosis is currently ineffective; therefore, elucidating the mechanism of liver fibrogenesis is of significant importance. Herein, the function and related mechanisms of lncRNA Snhg12 within hepatic fibrosis were investigated. Snhg12 expression was shown to be increased in mouse hepatic fibrotic tissue samples, and Snhg12 knockdown suppressed hepatic pathological injury and down-regulated the expression levels of fibrosis-associated proteins. Mechanistically, Snhg12 played a role in the early activation of mouse hepatic stellate cells (mHSCs) based on bioinformatics analysis, and Snhg12 was positively correlated with Igfbp3 expression. Further experimental results demonstrated that Snhg12 knockdown impeded mHSCs proliferation and activation and also downregulated the protein expression of Igfbp3. Snhg12 could interact with IGFBP3 and boost its protein stability, and overexpression of Igfbp3 partially reversed the inhibition of mHSCsproliferation and activation by the knockdown of Snhg12. In conclusion, LncRNA Snhg12 mediates liver fibrosis by targeting IGFBP3 and promoting its protein stability, thereby promoting mHSC proliferation and activation. Snhg12 has been identified as an underlying target for treating liver fibrosis.

The CRL3KCTD10 ubiquitin ligase-USP18 axis coordinately regulates cystine uptake and ferroptosis by modulating SLC7A11.

SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.

Targeting the protein folding transition state by mutation: Large scale (un)folding rate accelerations without altering native stability.

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.

Targeting ubiquitination machinery in cystic fibrosis: Where do we stand?

Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin-proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.

Utilization of low-stability variants in protein evolutionary engineering.

Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step. The esterase from the thermophilic bacterium Alicyclobacillus acidocaldarius was used as a model protein, and its activity at its optimum temperature of 65 °C was improved by evolutionary experiments using random mutations by error-prone PCR. After restabilization using low-stability variants with low-temperature (37 °C) activity, several re-stabilizing variants were obtained from a large number of variant libraries. Some of the restabilized variants achieved by removing the destabilizing mutations showed higher activity than that of the wild-type protein. This implies that low-stability variants with low-temperature activity can be re-evolved for future use. This method will enable further diversification of evolutionary pathways.

Cellular and epigenetic perspective of protein stability and its implications in the biological system.

Protein stability is a fundamental prerequisite in both experimental and therapeutic applications. Current advancements in high throughput experimental techniques and functional ontology approaches have elucidated that impairment in the structure and stability of proteins is intricately associated with the cause and cure of several diseases. Therefore, it is paramount to deeply understand the physical and molecular confounding factors governing the stability of proteins. In this review article, we comprehensively investigated the evolution of protein stability, examining its emergence over time, its relationship with organizational aspects and the experimental methods used to understand it. Furthermore, we have also emphasized the role of Epigenetics and its interplay with post-translational modifications (PTMs) in regulating the stability of proteins.

Proteins are essential for life and are used in many medical treatments. Understanding what makes proteins stable can help us use them more effectively. This review looks at how different things like temperature and pH affect protein stability. It also discusses how chemical changes in cells, called epigenetic modifications, can impact protein stability. Understanding these factors can help us develop better treatments and therapies.

USP11 modulates mitotic progression and senescence by regulating the p53-p21 axis through MDM2 deubiquitination.

USP11 is overexpressed in colorectal cancer (CRC) and breast cancer tissues compared to normal tissues, suggesting a role in promoting cell proliferation and inhibiting cell death. In this study, we observed that depleting USP11 inhibits cell proliferation and delays cell cycle progression. This depletion leads to increased p53 protein levels due to an extended half-life, resulting in elevated p21 mRNA levels in a p53-dependent manner. The rise in p53 protein upon USP11 depletion is linked to a reduced half-life of MDM2, a known E3 ligase for p53, via enhanced polyubiquitination of MDM2. These findings indicate that USP11 might act as a deubiquitinase for MDM2, regulating the MDM2-p53-p21 axis. Additionally, USP11 depletion promotes the induction of senescent cells in a manner dependent on its deubiquitinase activity. Our findings provide insights into the physiological significance of high USP11 expression in primary tumors and its reduction in senescent cells, highlighting its potential as a therapeutic target.

Pepper SUMO protease CaDeSI2 positively modulates the drought responses via deSUMOylation of clade A PP2C CaAITP1.

Posttranslational modification of multiple ABA signaling components is an essential process for the adaptation and survival of plants under stress conditions. In our previous study, we established that the pepper group A PP2C protein CaAITP1, one of the core components of ABA signaling, undergoes ubiquitination mediated by the RING-type E3 ligase CaAIRE1. In this study, we discovered an additional form of regulation mediated via the SUMOylation of CaAITP1. Pepper plants subjected to drought stress were characterized by reductions in both the stability and SUMOylation of CaAITP1 protein. Moreover, we identified a SUMO protease, Capsicum annuum DeSUMOylating Isopeptidase 2 (CaDeSI2), as a new interacting partner of CaAITP1. In vitro and in vivo analyses revealed that CaAITP1 is deSUMOylated by CaDeSI2. Silencing of CaDeSI2 in pepper plants led to drought-hypersensitive and ABA-hyposensitive phenotypes, whereas overexpression of CaDeSI2 in transgenic Arabidopsis plants resulted in the opposite phenotypes. Importantly, we found that the CaAITP1 protein was stabilized in response to the silencing of CaDeSI2, and CaDeSI2 and CaAITP1 co-silenced pepper plants were characterized by drought-tolerant phenotypes similar to those observed in CaAITP1-silenced pepper. Collectively, our findings indicate that CaDeSI2 reduces the stability of CaAITP1 via deSUMOylation, thereby positively regulating drought tolerance.

Mutations in the Receptor Binding Domain of Severe Acute Respiratory Coronavirus-2 Omicron Variant Spike Protein Significantly Stabilizes Its Conformation.

The Omicron variant and its sub-lineages are the only current circulating SARS-CoV-2 viruses worldwide. In this study, the conformational stability of the isolated Receptor Binding Domain (RBD) of Omicron's spike protein is examined in detail. The parent Omicron lineage has over ten mutations in the ACE2 binding region of the RBD that are specifically associated with its ß hairpin loop domain. It is demonstrated through biophysical molecular computations that the mutations in the ß hairpin loop domain significantly increase the intra-protein interaction energies of intra-loop and loop-RBD interactions. The interaction energy increases include the formation of new hydrogen bonds in the ß hairpin loop domain that help stabilize this critical ACE2 binding region. Our results also agree with recent experiments on the stability of Omicron's core ß barrel domain, outside of its loop domain, and help demonstrate the overall conformational stability of the Omicron RBD. It is further shown here through dynamic simulations that the unbound state of the Omicron RBD remains closely aligned with the bound state configuration, which was not observed for the wild-type RBD. Overall, these studies demonstrate the significantly increased conformational stability of Omicron over its wild-type configuration and raise a number of questions on whether conformational stability could be a positive selection feature of SARS-CoV-2 viral mutational changes.

Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry-state protein storage.

Protein biotherapeutics typically require expensive cold-chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold-chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., "cryoprotection" and "lyoprotection"), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake-forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1-16) were extracted via ethanol precipitation. PG size was generally consistent at ~70-100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1-16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, ß-galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate-buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein-PG13 mixtures had a cake-like appearance and all frozen protein-PG13 mixtures had a Tg' of ~ -26°C. The lyoprotection and cake-forming properties of PG13 were density-dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Regulation of PPARγ2 Stability and Activity by SHP-1.

The protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) plays an important role in modulating glucose and lipid homeostasis. We previously suggested a potential role of SHP-1 in the regulation of peroxisome proliferator-activated receptor γ2 (PPARγ2) expression and activity but the mechanisms were unexplored. PPARγ2 is the master regulator of adipogenesis, but how its activity is regulated by tyrosine phosphorylation is largely unknown. Here, we found that SHP-1 binds to PPARγ2 primarily via its N-terminal SH2-domain. We confirmed the phosphorylation of PPARγ2 on tyrosine-residue 78 (Y78), which was reduced by SHP-1 in vitro resulting in decreased PPARγ2 stability. Loss of SHP-1 led to elevated, agonist-induced expression of the classical PPARγ2 targets FABP4 and CD36, concomitant with increased lipid content in cells expressing PPARγ2, an effect blunted by abrogation of PPARγ2 phosphorylation. Collectively, we discovered that SHP-1 affects the stability of PPARγ2 through dephosphorylation thereby influencing adipogenesis.

Improved prediction of site-rates from structure with averaging across homologs.

Variation in mutation rates at sites in proteins can largely be understood by the constraint that proteins must fold into stable structures. Models that calculate site-specific rates based on protein structure and a thermodynamic stability model have shown a significant but modest ability to predict empirical site-specific rates calculated from sequence. Models that use detailed atomistic models of protein energetics do not outperform simpler approaches using packing density. We demonstrate that a fundamental reason for this is that empirical site-specific rates are the result of the average effect of many different microenvironments in a phylogeny. By analyzing the results of evolutionary dynamics simulations, we show how averaging site-specific rates across many extant protein structures can lead to correct recovery of site-rate prediction. This result is also demonstrated in natural protein sequences and experimental structures. Using predicted structures, we demonstrate that atomistic models can improve upon contact density metrics in predicting site-specific rates from a structure. The results give fundamental insights into the factors governing the distribution of site-specific rates in protein families.

Assessing the functional roles of coevolving PHD finger residues.

Although in silico folding based on coevolving residue constraints in the deep-learning era has transformed protein structure prediction, the contributions of coevolving residues to protein folding, stability, and other functions in physical contexts remain to be clarified and experimentally validated. Herein, the PHD finger module, a well-known histone reader with distinct subtypes containing subtype-specific coevolving residues, was used as a model to experimentally assess the contributions of coevolving residues and to clarify their specific roles. The results of the assessment, including proteolysis and thermal unfolding of wildtype and mutant proteins, suggested that coevolving residues have varying contributions, despite their large in silico constraints. Residue positions with large constraints were found to contribute to stability in one subtype but not others. Computational sequence design and generative model-based energy estimates of individual structures were also implemented to complement the experimental assessment. Sequence design and energy estimates distinguish coevolving residues that contribute to folding from those that do not. The results of proteolytic analysis of mutations at positions contributing to folding were consistent with those suggested by sequence design and energy estimation. Thus, we report a comprehensive assessment of the contributions of coevolving residues, as well as a strategy based on a combination of approaches that should enable detailed understanding of the residue contributions in other large protein families.

Stability of Protein Pharmaceuticals: Recent Advances.

There have been significant advances in the formulation and stabilization of proteins in the liquid state over the past years since our previous review. Our mechanistic understanding of protein-excipient interactions has increased, allowing one to develop formulations in a more rational fashion. The field has moved towards more complex and challenging formulations, such as high concentration formulations to allow for subcutaneous administration and co-formulation. While much of the published work has focused on mAbs, the principles appear to apply to any therapeutic protein, although mAbs clearly have some distinctive features. In this review, we first discuss chemical degradation reactions. This is followed by a section on physical instability issues. Then, more specific topics are addressed: instability induced by interactions with interfaces, predictive methods for physical stability and interplay between chemical and physical instability. The final parts are devoted to discussions how all the above impacts (co-)formulation strategies, in particular for high protein concentration solutions.'

Visible light triggers the formation of reactive oxygen species in monoclonal antibody formulations.

Most monoclonal antibody formulations require the presence of a surfactant, such as polysorbate, to ensure protein stability. The presence of high concentrations of polysorbate have been shown to enhance photooxidation of certain protein drug products when exposed to visible light. The current literature, however, suggest that photooxidation of polysorbate only occurs when exposed to visible light in combination with UVA light. This is probable as peroxides present in polysorbate solutions can be cleaved homolytically in the UVA region. In the visible region, photooxidation is not expected to occur as cleavage of peroxides is not expected at these wavelengths. This report presents findings suggesting that the presence of one or more photosensitiser(s) in polysorbate must be a cause and is required to catalyse the aerobic oxidation of polysorbate solutions upon exposure to visible light. Our investigation aimed to clarify the mechanism(s) of polysorbate photooxidation and explore the kinetics and the identity of the generated radicals and their impact on monoclonal antibody (mAb) degradation. Our study reveals that when polysorbate solutions are exposed to visible light between 400---800 nm in the absence of proteins, discoloration, radical formation, and oxygen depletion occur. We discuss the initial formation of reactive species, most likely occurring directly after reaction of molecular oxygen, with the presence of a triplet state photosensitizer, which is generated by intersystem crossing of the excited singlet state, leading predominantly to the formation of reactive species such as singlet oxygen species. When comparing the photooxidation of PS20 and PS80 in varying quality grades, we propose that singlet oxygen possesses potential for reacting with unsaturated fatty acids in PS80HP, however, PS20HP itself exhibited no measurable oxidation under the tested conditions. The study's final part delves into the photooxidation behaviour of different PS grades, examining its influence on the integrity of a mAb in the formulation. Finally, we examined the effect of photooxidation on the integrity of monoclonal antibodies. Our findings show that the exposure to visible light in polysorbate-containing mAb solutions at high PS concentrations of 4 mg∙ml-1 results in increased monoclonal antibody degradation, highlighting the need for cautious evaluation of the correct PS concentration to stabilise protein therapeutics.

Intracellular Protective Functions and Therapeutical Potential of Trehalose.

Trehalose is a naturally occurring, non-reducing saccharide widely distributed in nature. Over the years, research on trehalose has revealed that this initially thought simple storage molecule is a multifunctional and multitasking compound protecting cells against various stress factors. This review presents data on the role of trehalose in maintaining cellular homeostasis under stress conditions and in the virulence of bacteria and fungi. Numerous studies have demonstrated that trehalose acts in the cell as an osmoprotectant, chemical chaperone, free radical scavenger, carbon source, virulence factor, and metabolic regulator. The increasingly researched medical and therapeutic applications of trehalose are also discussed.

Studying protein stability in crowded environments by NMR.

Most proteins perform their functions in crowded and complex cellular environments where weak interactions are ubiquitous between biomolecules. These complex environments can modulate the protein folding energy landscape and hence affect protein stability. NMR is a nondestructive and effective method to quantify the kinetics and equilibrium thermodynamic stability of proteins at an atomic level within crowded environments and living cells. Here, we review NMR methods that can be used to measure protein stability, as well as findings of studies on protein stability in crowded environments mimicked by polymer and protein crowders and in living cells. The important effects of chemical interactions on protein stability are highlighted and compared to spatial excluded volume effects.

Grp78 destabilization of infectious prions is strain-specific and modified by multiple factors including accessory chaperones and pH.

Lethal neurodegenerative prion diseases result from the continuous accumulation of infectious and variably protease-resistant prion protein aggregates (PrPD) which are misfolded forms of the normally detergent soluble and protease-sensitive cellular prion protein. Molecular chaperones like Grp78 have been found to reduce the accumulation of PrPD, but how different cellular environments and other chaperones influence the ability of Grp78 to modify PrPD is poorly understood. In this work, we investigated how pH and protease-mediated structural changes in PrPD from two mouse-adapted scrapie prion strains, 22L and 87V, influenced processing by Grp78 in the presence or absence of chaperones Hsp90, DnaJC1, and Stip1. We developed a cell-free in vitro system to monitor chaperone-mediated structural changes to, and disaggregation of, PrPD. For both strains, Grp78 was most effective at structurally altering PrPD at low pH, especially when additional chaperones were present. While Grp78, DnaJC1, Stip1, and Hsp90 were unable to disaggregate the majority of PrPD from either strain, pretreatment of PrPD with proteases increased disaggregation of 22L PrPD compared to 87V, indicating strain-specific differences in aggregate structure were impacting chaperone activity. Hsp90 also induced structural changes in 87V PrPD as indicated by an increase in the susceptibility of its n-terminus to proteases. Our data suggest that, while chaperones like Grp78, DnaJC1, Stip1, and Hsp90 disaggregate only a small fraction of PrPD, they may still facilitate its clearance by altering aggregate structure and sensitizing PrPD to proteases in a strain and pH-dependent manner.

A small molecule compound 759 inhibits the wnt/beta-catenin signaling pathway via increasing the Axin protein stability.

Wnt/ß-catenin signaling plays important role in cancers. Compound 759 is one of the compounds previously screened to identify inhibitors of the Wnt/ß-catenin pathway in A549 cells [Lee et al. in Bioorg Med Chem Lett 20:5900-5904, 2010]. However, the mechanism by which Compound 759 induces the inhibition of the Wnt/ß-catenin pathway remains unknown. In our study, we employed various assays to comprehensively evaluate the effects of Compound 759 on lung cancer cells. Our results demonstrated that Compound 759 significantly suppressed cell proliferation and Wnt3a-induced Topflash activity and arrested the cell cycle at the G1 stage. Changes in Wnt/ß-catenin signaling-related protein expression, gene activity, and protein stability including Axin, and p21, were achieved through western blot and qRT-PCR analysis. Compound 759 treatment upregulated the mRNA level of p21 and increased Axin protein levels without altering the mRNA expression in A549 cells. Co-treatment of Wnt3a and varying doses of Compound 759 dose-dependently increased the amounts of Axin1 in the cytosol and inhibited ß-catenin translocation into the nucleus. Moreover, Compound 759 reduced tumor size and weight in the A549 cell-induced tumor growth in the in vivo tumor xenograft mouse model. Our findings indicate that Compound 759 exhibits potential anti-cancer activity by inhibiting the Wnt/ß-catenin signaling pathway through the increase of Axin1 protein stability.