Mechanical Stability Of Proteins Research Articles

Colyophilized Sugar-Polymer Dispersions for Enhanced Processing and Storage Stability.

Sucrose and trehalose pharmaceutical excipients are employed to stabilize protein therapeutics in a dried state. The mechanism of therapeutic protein stabilization is dependent on the sugars being present in an amorphous solid-state. Colyophilization of sugars with high glass transition polymers, polyvinylpyrrolidone (PVP), and poly(vinylpyrrolidone vinyl acetate) (PVPVA), enhances amorphous sugar stability. This study investigates the stability of colyophilized sugar-polymer systems in the frozen solution state, dried state postlyophilization, and upon exposure to elevated humidity. Binary systems of sucrose or trehalose with PVP or PVPVA were lyophilized with sugar/polymer ratios ranging from 2:8 to 8:2. Frozen sugar-PVPVA solutions exhibited a higher glass transition temperature of the maximally freeze-concentrated amorphous phase (Tg') compared to sugar-PVP solutions, despite the glass transition temperature (Tg) of PVPVA being lower than PVP. Tg values of all colyophilized systems were in a similar temperature range irrespective of polymer type. Greater hydrogen bonding between sugars and PVP and the lower hygroscopicity of PVPVA influenced polymer antiplasticization effects and the plasticization effects of residual water. Plasticization due to water sorption was investigated in a dynamic vapor sorption humidity ramping experiment. Lyophilized sucrose systems exhibited increased amorphous stability compared to trehalose upon exposure to the humidity. Recrystallization of trehalose was observed and stabilized by polymer addition. Lower concentrations of PVP inhibited trehalose recrystallization compared to PVPVA. These stabilizing effects were attributed to the increased hydrogen bonding between trehalose and PVP compared to trehalose and PVPVA. Overall, the study demonstrated how differences in polymer hygroscopicity and hydrogen bonding with sugars influence the stability of colyophilized amorphous dispersions. These insights into excipient solid-state stability are relevant to the development of stabilized biopharmaceutical solid-state formulations.

Comparison of Sucrose and Trehalose for Protein Stabilization Using Differential Scanning Calorimetry.

The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to explain this, the stabilization mechanism is still far from being fully understood. This study compares the stabilizing properties of trehalose with those of the structurally similar disaccharide sucrose. The stability has been evaluated for the two proteins, lysozyme and myoglobin, at both low and high temperatures by determining the glass transition temperature, Tg, and the denaturation temperature, Tden. The results show that the sucrose-containing samples exhibit higher Tden than the corresponding trehalose-containing samples, particularly at low water contents. The better stabilizing effect of sucrose at high temperatures may be explained by the fact that sucrose, to a greater extent, binds directly to the protein surface compared to trehalose. Both sugars show Tden elevation with an increasing sugar-to-protein ratio, which allows for a more complete sugar shell around the protein molecules. Finally, no synergistic effects were found by combining trehalose and sucrose. Conclusively, the exact mechanism of protein stabilization may vary with the temperature, as influenced by temperature-dependent interactions between the protein, sugar, and water. This variability can make trehalose to a superior stabilizer under some conditions and sucrose under others.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods, may have important implications in drug development and discovery. Here, we present the first approach that employs computational High-Throughput Virtual Screening (HTVS) and Molecular Docking to search for small-molecules in chemical libraries that function as mechano-regulators of the stability of human CD4, receptor of HIV-1. Using single-molecule force spectroscopy we prove that these small-molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, i.e., mechano-regulation, paving the way towards mechanopharmacology.

Polysome-bound mRNAs and translational mechanisms regulate drought tolerance in rice

Plants evolved several acquired tolerance traits for drought stress adaptation to maintain the cellular homeostasis. Drought stress at the anthesis stage in rice affects productivity due to the inefficiency of protein synthesis machinery. The effect of translational mechanisms on different pathways involved in cellular tolerance plays an important role. We report differential responses of translation-associated mechanisms in rice using polysome bound mRNA sequencing at anthesis stage drought stress in resistant Apo and sensitive IR64 genotypes. Apo maintained higher polysomes with 60 S-to-40 S and polysome-to-monosome ratios which directly correlate with protein levels under stress. IR64 has less protein levels under stress due to defective translation machinery and reduced water potential. Many polysome-bound long non-coding RNAs (lncRNA) were identified in both genotypes under drought, influencing translation. Apo had higher levels of N6-Methyladenosine (m6A) mRNA modifications that contributed for sustained translation. Translation machinery in Apo could maintain higher levels of photosynthetic machinery-associated proteins in drought stress, which maintain gas exchange, photosynthesis and yield under stress. The protein stability and ribosome biogenesis mechanisms favoured improved translation in Apo. The phytohormone signalling and transcriptional responses were severely affected in IR64. Our results demonstrate that, the higher translation ability of Apo favours maintenance of photosynthesis and physiological responses that are required for drought stress adaptation.

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species.

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.

Hypoxia-inducible factor 1 alpha protein increases without changes in mRNA during acute hypoxic exposure of the Gulf killifish, Fundulus grandis.

The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses of animals to low oxygen. While the hypoxia-responsiveness of HIF1 is generally attributed to the stabilization of the alpha protein subunit (HIF1α) at low oxygen, several studies on fish report increased tissue levels of HIF1A mRNA during hypoxia, suggesting transcriptional regulation. In the current study, HIF1α protein and HIF1A mRNA were determined in parallel in tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2l-1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1A mRNA levels were unaffected by hypoxia in any tissue. Moreover, HIF1α protein and HIF1A mRNA levels in the same tissues were not correlated with one another during either normoxia or hypoxia. Hence, an increase in HIF1α protein does not depend upon an increase in HIF1A mRNA during acute exposure to low oxygen in this species. The results support the widely accepted mechanism of post-translational protein stabilization, rather than new transcription, during the initial response of fish to hypoxia.

The OX40-TRAF6 axis promotes CTLA-4 degradation to augment antitumor CD8+ T-cell immunity.

Immune checkpoint blockade (ICB), including anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4), benefits only a limited number of patients with cancer. Understanding the in-depth regulatory mechanism of CTLA-4 protein stability and its functional significance may help identify ICB resistance mechanisms and assist in the development of novel immunotherapeutic modalities to improve ICB efficacy. Here, we identified that TNF receptor-associated factor 6 (TRAF6) mediates Lys63-linked ubiquitination and subsequent lysosomal degradation of CTLA-4. Moreover, by using TRAF6-deficient mice and retroviral overexpression experiments, we demonstrated that TRAF6 promotes CTLA-4 degradation in a T-cell-intrinsic manner, which is dependent on the RING domain of TRAF6. This intrinsic regulatory mechanism contributes to CD8+ T-cell-mediated antitumor immunity in vivo. Additionally, by using an OX40 agonist, we demonstrated that the OX40-TRAF6 axis is responsible for CTLA-4 degradation, thereby controlling antitumor immunity in both tumor-bearing mice and patients with cancer. Overall, our findings demonstrate that the OX40-TRAF6 axis promotes CTLA-4 degradation and is a potential therapeutic target for the improvement of T-cell-based immunotherapies.

CCNB1IP1 prevents ubiquitination-mediated destabilization of MYCN and potentiates tumourigenesis of MYCN-amplificated neuroblastoma.

MYCN amplification as a common genetic alteration that correlates with a poor prognosis for neuroblastoma (NB) patients. However, given the challenge of directly targeting MYCN, indirect strategies to modulate MYCN by interfering with its cofactors are attractive in NB treatment. Although cyclin B1 interacting protein 1 (CCNB1IP1) has been found to be upregulated in MYCN-driven mouse NB tissues, its regulation with MYCN and collaboration in driving the biological behaviour of NB remains unknown. To evaluate the expression and clinical significance of CCNB1IP1 in NB patients, public datasets, clinical NB samples and cell lines were explored. MTT, EdU incorporation, colony and tumour sphere formation assays, and a mouse xenograft tumour model were utilized to examine the biological function of CCNB1IP1. The reciprocal manipulation of CCNB1IP1 and MYCN and the underlying mechanisms involved were investigated by gain- and loss-of-function approaches, dual-luciferase assay, chromatin immunoprecipitation (CHIP) and co-immunoprecipitation (Co-IP) experiments. CCNB1IP1 was upregulated in MYCN-amplified (MYCN-AM) NB cell lines and patients-derived tumour tissues, which was associated with poor prognosis. Phenotypic studies revealed that CCNB1IP1 facilitated the proliferation and tumourigenicity of NB cells in cooperation with MYCN in vitro and in vivo. Mechanistically, MYCN directly mediates the transcription of CCNB1IP1, which in turn attenuated the ubiquitination and degradation of MYCN protein, thus enhancing CCNB1IP1-MYCN cooperativity. Moreover, CCNB1IP1 competed with F box/WD-40 domain protein 7 (FBXW7) for MYCN binding and enabled MYCN-mediated tumourigenesis in a C-terminal domain-dependent manner. Our study revealed a previously uncharacterized mechanism of CCNB1IP1-mediated MYCN protein stability and will provide new prospects for precise treatment of MYCN-AM NB based on MYCN-CCNB1IP1 interaction.

AIOLOS haploinsufficiency is associated with immunodeficiency, autoimmunity, and allergy

AIOLOS is a transcription factor of the IKAROS family, that is highly expressed in the lymphoid lineage and plays an important role in lymphocyte differentiation and development. Recently, two heterozygous AIOLOS germline mutations (G159R and N160S) were reported in primary immunodeficiency patients. Both variants associated distinctive and unique T and B cell differentiation defects, hematologic malignancies, and infectious disease susceptibility patterns through different dominant negative (DN) mechanisms. Here we report seven individuals from three unrelated families carrying AIOLOS variants Q402* (nonsense, 1 family) and E82K (missense, 2 families), leading to AIOLOS haploinsufficiency through different mechanisms of action. Clinically, patients with AIOLOS haploinsufficiency showed hypogammaglobulinemia, recurrent respiratory tract infections, and immune dysregulation manifested by autoimmunity and allergy, with incomplete clinical penetrance. Mechanistically, while the nonsense mutant Q402* is devoid of its C-terminal zinc-finger (ZF) dimerization domain (ZF5-6), it is still able to homodimerize with wildtype (WT) AIOLOS although unable to heterodimerize with WT IKAROS or HELIOS. We introduce new insights into AIOLOS biology as we found that AIOLOS ZF1, a part of AIOLOS DNA binding domain (ZF1-4), was associated with noncanonical dimerization and negative regulation of DNA binding. Further studies revealed that the mutant Q402* displayed abnormalities of DNA binding, pericentromeric targeting, post-transcriptional modification, and transcriptome regulation by haploinsufficiency. In contrast, mutant E82K showed overall normal AIOLOS functions; however, by affecting a newly defined AIOLOS protein stability domain, it also led to AIOLOS haploinsufficiency. Unlike the previously reported AIOLOS mutations, heterozygous variants Q402* and E82K do not have a DN effect over other IKAROS family proteins. Our data extend the spectrum of pathogenic variants of AIOLOS-associated diseases and provide a novel mechanism of homodimerization and protein stability that, when disrupted, could affect human health.

Mechanism of protein stabilization in crowded solution and preservation in desiccated sugar glass.

The highly crowded cellular milieu includes countless different macromolecules and osmolytes aimed at regulating osmotic stress while protecting proteins and other macromolecules. Of special interest is the formation of cellular glassy matrices composed of sugars, which strongly correlate with the ability of some yeast, nematodes, and tardigrades to survive desiccation and extreme temperature changes. Remarkably, these carbohydrates stabilize proteins in crowded solutions and preserve them in the dry glassy state. Yet, although considerable efforts have been directed to discern how crowding stabilizes proteins, the molecular-level stabilization mechanism has remained elusive. We have followed the effect of various osmolytes on the native state stability of model miniproteins in crowded solutions using circular dichroism (CD) spectroscopy and our newly developed mean-field theory for crowding. We show that protein native state stability and folding thermodynamic signature can be explained using three distinct types of interactions: excluded volume, solvent-osmolyte non-ideal mixing, and osmolyte-protein soft-interactions. Furthermore, using our combination of synchrotron radiation CD spectroscopy together with sampling enhanced molecular dynamics (MD) simulations we have been able to shed light on the stability and new emerging nanostructures of proteins when they are embedded in sugar glass.

Osmolyte induced protein stabilization: modulation of associated water dynamics might be a key factor.

The mechanism of protein stabilization by osmolytes remains one of the most important and long-standing puzzles. The traditional explanation of osmolyte-induced stability through the preferential exclusion of osmolytes from the protein surface has been seriously challenged by the observations like the concentration-dependent reversal of osmolyte-induced stabilization/destabilization. The more modern explanation of protein stabilization/destabilization by osmolytes considers an indirect effect due to osmolyte-induced distortion of the water structure. It provides a general mechanism, but there are numerous examples of protein-specific effects, i.e., a particular osmolyte might stabilize one protein, but destabilize the other, that could not be rationalized through such an explanation. Herein, we hypothesized that osmolyte-induced modulation of associated water might be a critical factor in controlling protein stability in such a medium. Taking different osmolytes and papain as a protein, we proved that our proposal could explain protein stability in osmolyte media. Stabilizing osmolytes rigidify associated water structures around the protein, whereas destabilizing osmolytes make them flexible. The strong correlation between the stability and the associated water dynamics, and the fact that such dynamics are very much protein specific, established the importance of considering the modulation of associated water structures in explaining the osmolyte-induced stabilization/destabilization of proteins. More interestingly, we took another protein, bromelain, for which a traditionally stabilizing osmolyte, sucrose, acts as a stabilizer at higher concentrations but as a destabilizer at lower concentrations. Our proposal successfully explains such observations, which is probably impossible by any known mechanisms. We believe this report will trigger much research in this area.

Climate and geology overwrite land use effects on soil organic nitrogen cycling on a continental scale

Abstract. Soil fertility and plant productivity are globally constrained by N availability. Proteins are the largest N reservoir in soils, and the cleavage of proteins into small peptides and amino acids has been shown to be the rate-limiting step in the terrestrial N cycle. However, we are still lacking a profound understanding of the environmental controls of this process. Here we show that integrated effects of climate and soil geochemistry drive protein cleavage across large scales. We measured gross protein depolymerization rates in mineral and organic soils sampled across a 4000 km long European transect covering a wide range of climates, geologies and land uses. Based on structural equation models we identified that soil organic N cycling was strongly controlled by substrate availability, e.g., by soil protein content. Soil geochemistry was a secondary predictor, by controlling protein stabilization mechanisms and protein availability. Precipitation was identified as the main climatic control on protein depolymerization, by affecting soil weathering and soil organic matter accumulation. In contrast, land use was a poor predictor of protein depolymerization. Our results highlight the need to consider geology and precipitation effects on soil geochemistry when estimating and predicting soil N cycling at large scales.

E3 ubiquitin ligase MID1 ubiquitinates and degrades type‐I interferon receptor 2

Type I interferon (IFN-I) is a common biological molecule used for the treatment of viral diseases. However, the clinical antiviral efficacy of IFN-I needs to be greatly improved. In this study, IFN-I receptor 2 (IFNAR2) was revealed to undergo degradation at the protein level in cells treated with IFN-I for long periods of time. Further studies found a physical interaction between the E3 ubiquitin ligase midline-1 (MID1) and IFNAR2. As a consequence, MID1 induced both K48- and K63-linked polyubiquitination of IFNAR2, which promoted IFNAR2 protein degradation in a lysosome-dependent manner. Conversely, knockdown of MID1 largely restricted IFN-I-induced degradation of IFNAR2. Importantly, MID1 regulated the strength of IFN-I signalling and IFN-I-induced antiviral activity. These findings reveal a regulatory mechanism of IFNAR2 ubiquitination and protein stability in IFN-I signalling, which could provide a potential target for improving the antiviral efficacy of IFN-I.

Modulation of human transthyretin stability by the mutations at histidine 88 studied by free energy simulation.

Human transthyretin (TTR) is a homotetrameric plasma protein associated with a high percentage of β-sheet, which forms amyloid fibrils and accumulates in tissues or extracellular matrix to cause amyloid diseases. Free energy simulations based on all-atom molecular dynamics simulations were carried out to analyze the effects of the His88 → Arg, Phe, and Tyr mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His → Arg, Phe, and Tyr mutations at position 88 are 6.48 ± 0.45, -9.99 ± 0.54, and 2.66 ± 0.33 kcal/mol, respectively. These calculated free energy change differences between wild type and the mutants are in excellent agreement with prior experimental values. Our simulation results show that the wild type of the TTR is more stable than H88R and H88Y mutants, whereas it is less stable than the H88F mutant. The free energy component analysis shows that the primary contribution to the free energy change difference (ΔΔG) for the His → Arg mutation arises from electrostatic interaction; the ΔΔG for the His → Phe mutation is from van der Waals and electrostatic interactions and that for the His → Tyr mutation from covalent interaction. The simulation results show that the free energy calculation with thermodynamic integration is beneficial for understanding the detailed microscopic mechanism of protein stability. The implications of the results for understanding stabilizing and destabilizing effect of the mutation and the contribution to protein stability are discussed.

Native Salt Bridges Are a Key Regulator of Ubiquitin's Mechanical Stability.

Although it is known that various intramolecular interactions determine protein mechanical stability, a detailed molecular-level understanding of the key regulators of protein mechanical stability is still lacking. Here, we present evidence for salt bridges in ubiquitin as important intramolecular interactions that can affect protein mechanical stability. Ubiquitin has two salt bridges: one relatively surface-exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Ubiquitin is a reversible post-translational modifier and is stable mechanically (Favgu = 185 pN). On breaking SB1, the mechanical stability of ubiquitin is slightly enhanced (Favgu = 193 pN). In contrast, the mechanical stability significantly decreased upon breaking SB2 (Favgu = 158 pN). These results suggest that SB1 are SB2 are regulators of the mechanical stability of ubiquitin. Interestingly, the mechanical stability decreased further (Favgu = 145 pN) for the double salt bridge (DB) null variant. Monte Carlo simulations elucidate that the main regulating factor is the spontaneous unfolding rate constant (ku0), being the highest for the DB null variant followed by the SB2 null variant, and it remains unaltered for the SB1 null variant, while the native-to-transition-state distance (xu) remains unchanged. Our study provides mechanistic understanding on how two native salt bridges can independently regulate the mechanical stability in a protein, which has implications in designing protein-based robust biomaterials in the future.

EMT-related transcription factors and protein stabilization mechanisms involvement in cadherin switch of head and neck squamous cell carcinoma

Epithelial to mesenchymal transition (EMT) describes a process where epithelial tumor cells acquire mesenchymal characteristics. EMT often correlates with invasion and an increased cell migration potential by losing cellular polarity and cell-cell junctions. It is mainly induced by tumor-microenvironment factors, such as TGF-beta 1 and IL-6, which activate the increased expression of the EMT-transcription factor (TF) Slug. We previously reported the Slug/Krüppel-like factor 4 (KLF4) switch in EMT in HNSCC, and found, that in human papilloma virus (HPV)-negative HNSCC Slug gene expression was significant higher represented, than in HPV-positive HNSCC.The purpose of this study was to investigate the impact of KLF4 and Slug on the regulation of the cadherin switch and on the EMT phenotype.Gene expression of KLF4 positive correlated with E-cadherin in 71 head and neck squamous cell carcinoma (HNSCC) patient tissue samples, which we also confirmed by the investigation of the Cancer Genome Atlas database (TCGA). HPV-transcripts contributed to stabilization of KLF4 at protein level, and simultaneously upregulated E-cadherin. Furthermore, ectopic KLF4 overexpression was associated with epithelial gene expression by induction of E-cadherin, β-catenin and 70-kDa heat shock protein (HSP-70). The presence of HSP-70 ensures the membranous localization of E-cadherin, therefore, the ability of cells to form cadherin/catenin complexes and cellular linkages.In conclusion, KLF4 is a major regulator of the epithelial cadherin-adhesion in HNSCC.

ERK1/2 inhibits Cullin 3/SPOP-mediated PrLZ ubiquitination and degradation to modulate prostate cancer progression

The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in prostate cancer (PCa), but how SPOP functions as a tumor suppressor and contributes to PCa pathogenesis remains poorly understood. Prostate Leucine Zipper (PrLZ) serves as a prostate-specific and androgen-responsive gene, which plays a pivotal role in the malignant progression of PCa. However, the upstream regulatory mechanism of PrLZ protein stability and its physiological contribution to PCa carcinogenesis remain largely elusive. Here we report that PrLZ can be degraded by SPOP. PrLZ abundance is elevated in SPOP-mutant expressing PCa cell lines and patient specimens. Meanwhile, ERK1/2 might regulate SPOP-mediated PrLZ degradation through phosphorylating PrLZ at Ser40, which blocks the interaction between SPOP and PrLZ. In addition, we identify IL-6 might act as an upstream PrLZ degradation regulator via promoting its phosphorylation by ERK1/2, leading to its impaired recognition by SPOP. Thus, our study reveals a novel SPOP substrate PrLZ which might be controlled by ERK1/2-mediated phosphorylation, thereby facilitating to explore novel drug targets and improve therapeutic strategy for PCa.

Suppression of the doxorubicin response by hypoxia-inducible factor-1α is strictly dependent on oxygen concentrations under hypoxic conditions

Hypoxia-inducible factor-1α (HIF-1α) and p53 are involved in anticancer drug resistance under hypoxic conditions. Here, we found that the cytotoxicity of anticancer drugs (doxorubicin, gemcitabine, and cisplatin) was lower at 1% O2 than at 5% O2. We examined the effects of these drugs on HIF-1α and p53 expression under different hypoxic oxygen concentrations. At 5% O2, the drugs decreased HIF-1α expression and increased p53 levels. At 1% O2, the drugs increased HIF-1α expression but did not alter p53 levels. When the HIF-1α protein was stabilized by DMOG under normoxic conditions, doxorubicin did not increase the level of p53 expression. These results show that the maintenance of HIF-1α expression blocked doxorubicin-dependent increases in p53 expression. We hypothesized the mechanism of HIF-1α protein translation might be different between at 5% and at 1% O2, because many reports indicate that the same mechanism of HIF-1α protein stabilization occurs under hypoxic conditions, such as 5% and 1% O2. The level of phosphorylated-4E-BP1, which causes translation of HIF-1α, was higher at 1% O2 than at 5% O2. Our results suggest that the sensitivity of tumor cells to anticancer drugs is dependent oxygen concentrations under hypoxic conditions, and involves 4E-BP1-dependent stabilization of the HIF-1α protein.

Impact of lyoprotectors on protein-protein separation in the solid state: Neutron- and X-ray-scattering investigation

BackgroundPolyhydroxycompounds (PHC) are used as lyoprotectors to minimize aggregation of pharmaceutical proteins during freeze-drying and storage. MethodsLysozyme/PHC mixtures with 1:1 and 1:3 (w/w) ratios are freeze-dried from either H2O or D2O solutions. Disaccharides (sucrose and trehalose), monosaccharide (glucose), and sugar alcohol (sorbitol) are used in the study. Small-angle neutron and X-ray scattering (SANS and SAXS) are applied to study protein-protein interaction in the freeze-dried samples. ResultsProtein interaction peak in the freeze-dried mixtures has been detected by both SANS (D2O-based samples only) and SAXS (both D2O- and H2O-based). In the 1:1 mixtures, protein separation distances are similar (center-of-mass distance of approx. 31 Å) between all lyoprotectors studied. Mixtures with a higher content of the disaccharides (1:3 ratio) have a higher separation distance of approx 40 Å. The higher separation could reduce protein-protein contacts and therefore be associated with less favourable aggregation conditions. In the 1:3 mixtures with glucose and sorbitol, complex SANS and SAXS/WAXS patterns are observed. The pattern for the glucose sample indicate two populations of lysozyme molecules, while the origin of multiple SAXS peaks in the lysozyme/sorbitol 1:3 mixture is uncertain. ConclusionsProtein-protein separation distance is determined predominantly by the lyoprotector/protein weight ratio. General significanceUse of SANS and SAXS improves understanding of mechanisms of protein stabilization by sugars in freeze-dried formulations, and provide a tool to verify hypothesis on relationship between protein/protein separation and aggregation propensity in the dried state.

Conformational Flexibility and Local Frustration in the Functional States of the SARS-CoV-2 Spike B.1.1.7 and B.1.351 Variants: Mutation-Induced Allosteric Modulation Mechanism of Functional Dynamics and Protein Stability.

Structural and functional studies of the SARS-CoV-2 spike proteins have recently determined distinct functional states of the B.1.1.7 and B.1.351 spike variants, providing a molecular framework for understanding the mechanisms that link the effect of mutations with the enhanced virus infectivity and transmissibility. A detailed dynamic and energetic analysis of these variants was undertaken in the present work to quantify the effects of different mutations on functional conformational changes and stability of the SARS-CoV-2 spike protein. We employed the efficient and accurate coarse-grained (CG) simulations of multiple functional states of the D614G mutant, B.1.1.7 and B.1.351 spike variants to characterize conformational dynamics of the SARS-CoV-2 spike proteins and identify dynamic signatures of the functional regions that regulate transitions between the closed and open forms. By combining molecular simulations with full atomistic reconstruction of the trajectories and the ensemble-based mutational frustration analysis, we characterized how the intrinsic flexibility of specific spike regions can control functional conformational changes required for binding with the host-cell receptor. Using the residue-based mutational scanning of protein stability, we determined protein stability hotspots and identified potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. The results suggested that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites and the inter-protomer hinges of functional motions. The proposed mechanism of mutation-induced energetic frustration may result in greater adaptability and the emergence of multiple conformational states in the open form. This study suggested that SARS-CoV-2 B.1.1.7 and B.1.351 variants may leverage the intrinsic plasticity of functional regions in the spike protein for mutation-induced modulation of protein dynamics and allosteric regulation to control binding with the host cell receptor.