Uncovering DUB Selectivity through an Ion Mobility-Based Assessment of Ubiquitin Chain Isomers.

Ubiquitination is a reversible post-translational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological functions. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native unlabeled Ub conjugates. Here, we report an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-mass spectrometry (IM-MS) can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM-MS is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single-component mixtures.

[Effects of branched-chain amino acid supplementation on skeletal muscle proteolytic pathways after exercise-induced muscle injury in rats].

OBJECTIVE: To explore the role of branched-chain amino acid(BCAA) supplementation on muscle damage and the regulation of Krüppel-like factor 15(KLF15) and nuclear factor kappa B(NF-κB) mediated proteolytic pathways after an acute eccentric exercise. METHODS: Male SD rats were divided into placebo group(PLA) and BCAA group(BCAA) randomly, 32 rats per group. Both group were then placed into subgroups: placebo and pre-exercise group(PC), placebo and immediately after exercise group(PE), placebo and 6 h after exercise group(PE6), placebo and 12 h after exercise group(PE12), BCAA and pre-exercise group(BC), BCAA and immediately after exercise group(BE), BCAA and 6 h after exercise group(BE6), BCAA and 12 h after exercise group(BE12), 8 rats per group. Rats in BCAA groups were supplied with BCAA(1 g/(kg·d·BW), 3 days) before the exercise day and placebo groups with equal volume of distilled water. The exercised groups performed a 2 h eccentric exercise on treadmill(16 m/min, -16° slope). Blood and gastrocnemius were collected according to the time points. RT-qPCR was used to measure the mRNA expression of KLF15, NF-κB, FoxO1, Atrogin-1 and MuRF1 in gastrocnemius. RESULTS: (1) No damage was found in myocytes of BC and PC group. The process of morphological damage in BCAA group was relatively faster. (2) The mRNA expression levels of KLF15, FoXO1, Atrogin-1 and MuRF1 in PE were higher than those in PC(P<0.05, P<0.01), NF-κB and Atrogin-1 in PE12 were higher than those in PC(P<0.05). The mRNA expression levels of FoXO1 in BE were higher than those in BC(P<0.05). Compared with PE, the mRNA expression levels of KLF15, Atrogin-1 and MuRF1 in BE were lower(P<0.05, P<0.01), NF-κB and Atrogin-1 in BE12 were lower than those in PE12(P<0.05). The level of serum 3-MH in PE12 group was higher than that in PC group(P<0.05). CONCLUSION: The proteolysis of skeletal muscle after high-intensity eccentric exercise is mediated by two different pathways: KLF15 and NF-κB, whose activation is time-dependent. BCAA may reduce skeletal muscle proteolysis by lowering the level of gene transcription in the KLF15 and NF-κB related protein degradation pathway, which occurs immediately after exercise.

Systematic Potency and Property Assessment of VHL Ligands and Implications on PROTAC Design.

Herein, we describe a systematic SAR- and SPR-investigation of the peptidomimetic hydroxy-proline based VHL-ligand VH032, from which most to-date published VHL-targeting PROTACs have been derived. This study provides for the first time a consistent data set which allows for direct comparison of structural variations including those which were so far hidden in patent literature. The gained knowledge about improved VHL binders was used to design a small library of highly potent BRD4-degraders comprising different VHL exit vectors. Newly designed degraders showed favorable molecular properties and significantly improved degradation potency compared to MZ1.

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

Invited review: Muscle protein breakdown and its assessment in periparturient dairy cows.

Mobilization of body reserves including fat, protein, and glycogen is necessary to overcome phases of negative nutrient balance typical for high-yielding dairy cows during the periparturient period. Skeletal muscle, the largest internal organ in mammals, plays a crucial role in maintaining metabolic homeostasis. However, unlike in liver and adipose tissue, the metabolic and regulatory role of skeletal muscle in the adaptation of dairy cows to the physiological needs of pregnancy and lactation has not been studied extensively. The functional integrity and quality of skeletal muscle are maintained through a constant turnover of protein, resulting from both protein breakdown and protein synthesis. Thus, muscle protein breakdown (MPB) and synthesis are intimately connected and tightly controlled to ensure proper protein homeostasis. Understanding the regulation of MPB, the catabolic component of muscle turnover, and its assessment are therefore important considerations to provide information about the timing and extent of tissue mobilization in periparturient dairy cows. Based on animal models and human studies, it is now evident that MPB occurs via the integration of 3 main systems: autophagy-lysosomal, calpain Ca2+-dependent cysteine proteases, and the ubiquitin-proteasome system. These 3 main systems are interconnected and do not work separately, and the regulation is complex. The ubiquitin-proteasomal system is the most well-known cellular proteolytic system and plays a fundamental role in muscle physiology. Complete degradation of a protein often requires a combination of the systems, depending on the physiological situation. Determination of MPB in dairy cows is technically challenging, resulting in a relative dearth of information. The methods for assessing MPB can be divided into either direct or indirect measurements, both having their strengths and limitations. Available information on the direct measures of MPB primarily comes from stable isotopic tracer methods and those of indirect measurements from assessing expression and activity measures of the components of the 3 MPB systems in muscle biopsy samples. Other indirect approaches (i.e., potential indicators of MPB), including ultrasound imaging and measuring metabolites from muscle degradation (i.e., 3-methylhistidine and creatinine), seem to be applicable methods and can provide useful information about the extent and timing of MPB. This review presents our current understanding, including methodological considerations, of the process of MPB in periparturient dairy cows.

Immunocytochemical Assessment of ACE2 and TMPRSS2 in Nasopharyngeal Swabs from SARS-CoV-2 Patients.

BACKGROUND: SARS-CoV-2 is a positive-sense single-stranded RNA virus. It is enveloped by four structural proteins. The entry of the virus into the host cells is mediated by spike protein binding to the angiotensin converting enzyme 2 (ACE2) and proteolytic cleavage by transmembrane protease serine 2 (TMPRSS2). In this study, we analyzed the expression of the ACE2 receptor and TMPRSS2 in cases under investigation for SARS-CoV-2 infection. METHODS: The study was carried out using the viral transport medium of consecutive nasopharyngeal swabs from 300 people under examination for SARS-CoV-2 infection. All samples underwent the SARS-CoV-2 transcriptase-mediated amplification assay (Procleix® SARS-CoV-2) to detect the virus. Immunocytochemistry was used in each sample to detect the presence of the SARS-CoV-2 nucleoprotein, the ACE2 receptor, and TMPRSS2. RESULTS: An immunocytochemical study with monoclonal antibody against SARS-CoV-2 viral nucleoprotein showed positivity in squamous cells. ACE2 were not detected in the squamous cells obtained from the nasopharyngeal samples. CONCLUSIONS: SARS-CoV-2 predominantly localizes to squamous cells in cytology samples of patients with positive transcriptase-mediated amplification SARS-CoV-2 assay results. The immunocytochemical negativity for ACE2 evidenced in the present study could be related to the cellular heterogeneity present in the nasopharyngeal smear samples and could be related to variations at the genomic level. Our results suggest that SARS-CoV-2 might be present in the nasopharyngeal region because viral cell junctions are weaker. This facilitates viral concentration, infective capacity and migration to specific organs, where SARS-CoV-2 infects target cells by binding to their receptors and then entering.

Peptide Backbone Modifications for the Assessment of Cleavage Site Relevance in Precursors of Signaling Peptides.

The physiological relevance of site-specific precursor processing for the biogenesis of peptide hormones and growth factors can be demonstrated in genetic complementation experiments, in which a gain of function is observed for the cleavable wild-type precursor, but not for a non-cleavable precursor mutant. Similarly, cleavable and non-cleavable synthetic peptides can be used in bioassays to test whether processing is required for bioactivity. In genetic complementation experiments, site-directed mutagenesis has to be used to mask a processing site against proteolysis. Peptide-based bioassays have the distinctive advantage that peptides can be protected against proteolytic cleavage by backbone modifications, i.e., without changing the amino acid sequence. Peptide backbone modifications have been employed to increase the metabolic stability of peptide drugs, and in basic research, to investigate whether processing at a certain site is required for precursor maturation and formation of the bioactive peptide. For this approach, it is important to show that modification of the peptide backbone has the desired effect and does indeed protect the respective peptide bond against proteolysis. This can be accomplished with the MALDI-TOF mass spectrometry-based assay we describe here.

Assessment of milk protein digestion kinetics: effects of denaturation by heat and protein type used.

Knowledge about how molecular properties of proteins affect their digestion kinetics is crucial to understand protein postprandial plasma amino acid (AA) responses. Previously it was found that a native whey protein isolate (NWPI) and heat denatured whey protein isolate (DWPI) elicit comparable postprandial plasma AA peak concentrations in neonatal piglets, while a protein base ingredient for infant formula (PBI, a ß-casein-native whey protein mixture) caused a 39% higher peak AA concentration than NWPI. We hypothesized that both whey protein denaturation by heat as well as changing protein composition by including ß-casein, increases the rate of intact protein loss, and that changing the protein composition (by including ß-casein), but not whey protein denaturation, yields a faster absorbable product release. Therefore NWPI (91% native), DWPI (91% denatured) and PBI hydrolysis was investigated in a semi-dynamic in vitro digestion model (SIM). NWPI and DWPI hydrolysis were also compared in a dynamic digestion model with dialysis (TIM-1) to exclude potential product inhibition effects that may occur in a closed vessel digestion model as SIM. In both models, the degree of hydrolysis (DH), loss of intact protein, and release of absorbable products (SIM: <0.5 kDa peptides and free AA, TIM-1: bioaccessible AA) were monitored. Additionally, in SIM, intermediate product amounts and their characteristics were determined. DWPI showed considerably faster intact protein loss, but similar DH and absorbable product release kinetics compared with NWPI in both models. Furthermore, more, relatively large, intermediate products were released from DWPI than from NWPI. PBI showed increased intact protein loss, similar DH, and absorbable product release kinetics, but more, relatively small, intermediate products than NWPI. In conclusion, both whey protein denaturation and ß-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during in vitro digestion. Our results suggest that intermediate digestion product characteristics are important in relation to postprandial AA responses.

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients.

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

Importance of Three-Body Problems and Protein-Protein Interactions in Proteolysis-Targeting Chimera Modeling: Insights from Molecular Dynamics Simulations.

Proteolysis-targeting chimeras (PROTACs) are a class of bifunctional molecules that can induce the ubiquitin degradation of its target protein by hijacking the E3 ligase to form a target protein-PROTAC-E3 ligase ternary complex. Its underlying principle has inspired the development of a wide range of protein degraders that are similar to or beyond PROTACs in recent years. The formation of the ternary complexes is the key to the success of PROTAC-induced protein degradation. Nevertheless, the lack of effective ternary complex modeling techniques has limited the application of computer-aided drug discovery tools to this emerging and fast developing new land in drug industry. Thus, in this study, we explored the application of the more physically sound molecular dynamics simulation and the molecular mechanics combined with the generalized Born and surface area continuum solvation (MM/GBSA) method to solve the underlying three-body problem in PROTAC modeling. We first verified the accuracy of our approach using a series of known Brd4 BD2 degraders. The calculated binding energy showed a good correlation with the experimental Kd values. The modeling of a unique property, namely, the α value, for PROTACs was also first and accurately performed to our best knowledge. The results also demonstrated the importance of PROTAC-induced protein-protein interactions in its modeling, either qualitatively or quantitatively. Finally, by standing on the success of earlier docking-based approaches, our protocol was also applied as a rescoring function in pose prediction. The results showed a notable improvement in reranking the initial poses generated from a modified Rosetta method, which was reportedly one of the best among a handful of PROTAC modeling approaches available in this field. We hope this work could provide a practical protocol and more insights to study the binding and the design of PROTACs and other protein degraders.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

BACKGROUND: Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS: Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION: Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

Keratinases as Versatile Enzymatic Tools for Sustainable Development.

To reduce anthropological pressure on the environment, the implementation of novel technologies in present and future economies is needed for sustainable development. The food industry, with dairy and meat production in particular, has a significant environmental impact. Global poultry production is one of the fastest-growing meat producing sectors and is connected with the generation of burdensome streams of manure, offal and feather waste. In 2020, the EU alone produced around 3.2 million tonnes of poultry feather waste composed primarily of keratin, a protein biopolymer resistant to conventional proteolytic enzymes. If not managed properly, keratin waste can significantly affect ecosystems, contributing to environmental pollution, and pose a serious hazard to human and livestock health. In this article, the application of keratinolytic enzymes and microorganisms for promising novel keratin waste management methods with generation of new value-added products, such as bioactive peptides, vitamins, prion decontamination agents and biomaterials were reviewed.

Design, expression and functional assessment of novel engineered serratiopeptidase analogs with enhanced protease activity and thermal stability.

Serratiopeptidase is a bacterial protease that has been used medicinally in variety of applications. Though, some drawbacks like sensitivity to environmental conditions and low penetration into cells limited its usage as a potent pharmaceutical agent. This study aimed to produce four novel truncated serratiopeptidase analogs with different lengths and possessing one disulfide bridge, in order to enhance protease activity and thermal stability of this enzyme. Mutagenesis and truncation were performed using specific primers by conventional and overlap PCR. The recombinant proteins were expressed in E. coli cells then purified and their protease activity and stability were checked at different pH and temperatures in comparison to the native form of the enzyme, Serra473. Enzyme activity assay showed that T306 [12-302 ss] was not further active which could be due to the large truncation. However, T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] proteins showed higher proteolytic activity comparing to Serra473. These analogs were active at temperatures of 25-90 °C and pH 6-9.5. Interestingly, remaining enzyme activity of T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] forms at 90 °C calculated as 87, 83 and 86 percent, respectively, comparing to the activity at room temperature. However, residual activity at the same conditions was 50% for the full length enzyme. Formation of disulfide bond in engineered serratiopeptidases could be the main reason for higher thermal stability compared to Serra473. Thermostability of T344 [8-339 ss], as the most thermostable designed serratiopeptidase, was additionally confirmed using differential scanning calorimetry.

Methods for Quantitative Assessment of Protein Degradation.

Assessment of small molecules that promote selective protein degradation (degraders) requires detailed characterization and measurement of protein levels in cells. Here we describe ratio-metric methods based on a dual fluorescent GFP/mCherry reporter system to quantify cellular protein levels. We further develop a kinetic framework for the analysis of such data. We describe two methods of generating the stable GFP-protein of interest (POI)/mCherry reporter cell lines, alternative readout methods by FACS and Laser Scanning Cytometry as well as the corresponding tools used for processing and analysis of such data. Finally, we show that the commonly used half-maximal degradation constant (DC50) or maximum degradation efficacy (Dmax) metrics are time-dependent and propose a time-invariant Michaelis-Menten-like analysis of degradation kinetics with analogous key parameters Km app and Vmax app.

MicroRNA-205-5p targets E2F1 to promote autophagy and inhibit pulmonary fibrosis in silicosis through impairing SKP2-mediated Beclin1 ubiquitination.

Silicosis is an occupational disease characterized by extensive pulmonary fibrosis, and the underlying pathological process remains uncertain. Herein, we explored the molecular mechanism by which microRNA-205-5p (miR-205-5p) affects the autophagy of alveolar macrophages (AMs) and pulmonary fibrosis in mice with silicosis through the E2F transcription factor 1 (E2F1)/S-phase kinase-associated protein 2 (SKP2)/Beclin1 axis. Alveolar macrophages (MH-S cells) were exposed to crystalline silica (CS) to develop an in vitro model, and mice were treated with CS to establish an in vivo model. Decreased Beclin1 and increased SKP2 and E2F1 were identified in mice with silicosis. We silenced or overexpressed miR-205-5p, E2F1, SKP2 and Beclin1 to investigate their potential roles in pulmonary fibrosis in vivo and autophagy in vitro. Recombinant adenovirus mRFP-GFP-LC3 was transduced into the MH-S cells to assay autophagic flow. Knocking down Beclin1 promoted pulmonary fibrosis and suppressed the autophagy. Co-immunoprecipitation and ubiquitination assays suggested that SKP2 induced K48-linked ubiquitination of Beclin1. Furthermore, chromatin immunoprecipitation-PCR revealed the site where E2F1 bound to the SKP2 promoter between 1638 bp and 1645 bp. As shown by dual-luciferase reporter gene assay, the transfection with miR-205-5p mimic inhibited the luciferase activity of the wild-type E2F1 3'untranslated region, suggesting that miR-205-5p targeted E2F1. Additionally, miR-205-5p overexpression increased autophagy and reduced the pulmonary fibrosis, while overexpression of E2F1 or SKP2 or inhibition of Beclin1 could annul this effect. The current study elucidated that miR-205-5p targeted E2F1, thereby inhibiting SKP2-mediated Beclin1 ubiquitination to promote macrophage autophagy and inhibit pulmonary fibrosis in mice with silicosis.

Signaling through polymerization and degradation: Analysis and simulations of T cell activation mediated by Bcl10.

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-κB. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional, super-resolution optical micrographs, we quantitatively characterize TCR-stimulated Bcl10 filament assembly and length dynamics, and demonstrate that filaments become shorter over time. Additionally, we develop an image-based, bootstrap-like resampling method that demonstrates the preferred association between autophagosomes and both Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, we demonstrate that the speed of filament disassembly preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes.

Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake.

TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.

A freeze-and-thaw-induced fragment of the microtubule-associated protein tau in rat brain extracts: implications for the biochemical assessment of neurotoxicity.

Tau is a microtubule-associated protein (MAP) responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In the present study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots of the same extracts were stored for at least 2 weeks at either -20 or -80°C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ∼25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry (MS) analysis in excised bands revealed this ∼25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80°C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

Targeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological Development.

Bromodomain and extraterminal (BET) proteins bind acetylated lysine residues in histones and nonhistone proteins via tandem bromodomains and regulate chromatin dynamics, cellular processes, and disease procession. Thus targeting BET proteins is a promising strategy for treating various diseases, especially malignant tumors and chronic inflammation. Many pan-BET small-molecule inhibitors have been described, and some of them are in clinical evaluation. Nevertheless, the limited clinical efficacy of the current BET inhibitors is also evident and has inspired the development of new technologies to improve their clinical outcomes and minimize unwanted side effects. In this Review, we summarize the latest protein characteristics and biological functions of BRD4 as an example of BET proteins, analyze the clinical development status and preclinical resistance mechanisms, and discuss recent advances in BRD4-selective inhibitors, dual-target BET inhibitors, proteolysis targeting chimera degraders, and protein-protein interaction inhibitors.