Myogenic microRNAs as Therapeutic Targets for Skeletal Muscle Mass Wasting in Breast Cancer Models.

Breast cancer is the type of cancer with the highest prevalence in women worldwide. Skeletal muscle atrophy is an important prognostic factor in women diagnosed with breast cancer. This atrophy stems from disrupted skeletal muscle homeostasis, triggered by diminished anabolic signalling and heightened inflammatory conditions, culminating in an upregulation of skeletal muscle proteolysis gene expression. The importance of delving into research on modulators of skeletal muscle atrophy, such as microRNAs (miRNAs), which play a crucial role in regulating cellular signalling pathways involved in skeletal muscle protein synthesis and degradation, has been recognised. This holds true for conditions of homeostasis as well as pathologies like cancer. However, the determination of specific miRNAs that modulate skeletal muscle atrophy in breast cancer conditions has not yet been explored. In this narrative review, we aim to identify miRNAs that could directly or indirectly influence skeletal muscle atrophy in breast cancer models to gain an updated perspective on potential therapeutic targets that could be modulated through resistance exercise training, aiming to mitigate the loss of skeletal muscle mass in breast cancer patients.

Alterations in Proteostasis Mechanisms in Niemann-Pick Type C Disease.

Niemann-Pick Type C (NPC) represents an autosomal recessive disorder with an incidence rate of 1 in 150,000 live births, classified within lysosomal storage diseases (LSDs). The abnormal accumulation of unesterified cholesterol characterizes the pathophysiology of NPC. This phenomenon is not unique to NPC, as analogous accumulations have also been observed in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. Interestingly, disturbances in the folding of the mutant protein NPC1 I1061T are accompanied by the aggregation of proteins such as hyperphosphorylated tau, α-synuclein, TDP-43, and ß-amyloid peptide. These accumulations suggest potential disruptions in proteostasis, a regulatory process encompassing four principal mechanisms: synthesis, folding, maintenance of folding, and protein degradation. The dysregulation of these processes leads to excessive accumulation of abnormal proteins that impair cell function and trigger cytotoxicity. This comprehensive review delineates reported alterations across proteostasis mechanisms in NPC, encompassing changes in processes from synthesis to degradation. Additionally, it discusses therapeutic interventions targeting pharmacological facets of proteostasis in NPC. Noteworthy among these interventions is valproic acid, a histone deacetylase inhibitor (HDACi) that modulates acetylation during NPC1 synthesis. In addition, various therapeutic options addressing protein folding modulation, such as abiraterone acetate, DHBP, calnexin, and arimoclomol, are examined. Additionally, treatments impeding NPC1 degradation, exemplified by bortezomib and MG132, are explored as potential strategies. This review consolidates current knowledge on proteostasis dysregulation in NPC and underscores the therapeutic landscape targeting diverse facets of this intricate process.

BacPROTACs targeting Clp protease: a promising strategy for anti-mycobacterial drug discovery.

Tuberculosis (TB) has claimed more lives over the course of two millennia than any other infectious disease worldwide. In 2021, the World Health Organization (WHO) estimated that 10.6 million people were diagnosed with TB, resulting in the deaths of 1.4 million HIV-negative individuals. The emergence of multidrug-resistant TB (MDR-TB), defined as resistance to at least rifampicin (RIF) and isoniazid (INH), and extensively drug-resistant TB (XDR-TB), poses the primary challenge to overcome in the coming years. We have recently conducted an extensive analysis of investments and research endeavours in the field, with the overarching objective of achieving the established milestone of TB eradication by the year 2030. Over the past several years, there has been notable progress in advancing a multitude of promising compounds, each possessing distinct mechanisms of action, into clinical phases of development. However, it is worth noting that strains of mycobacteria resistant to current antitubercular drugs have already emerged for some of these compounds The exploration of the innovative Proteolytic Target Chimeras (PROTACs) protein degradation approach has emerged as a viable avenue for the discovery of novel antimicrobials. While the ubiquitin system is exclusive to eukaryotic cells, certain bacteria use a similar degradation system that relies on the recognition of phosphorylated arginine residues (pArg) by the ClpC:ClpP (ClpCP) protease, thereby leading to protein degradation. In this opinion article, we have described and analized the advances in the use of PROTACs that leverage bacterial proteolytic machinery (BacPROTACs) to design new antitubercular agents. Scope Statement. The development of novel pharmaceuticals for tuberculosis treatment is deemed urgently necessary due to the emergence of resistant strains. In this context, the introduction of new technologies capable of alleviating the disease and attaining the objectives outlined by the World Health Organization is imperative. Among the innovative strategies, the degradation of proteins that are crucial for the survival of the bacillus holds promise for generating new medications, particularly those that are effective at treating latent (non-replicating) Mycobacterium tuberculosis. Within this perspective, we present the advancements and obstacles encountered in the exploration of new BacPROTAC compounds, with the intention of encouraging research and illuminating challenges associated with the implementation of BacPROTACs to address to the global tuberculosis crisis.

Differential expression of PSMC4, SKP1, and HSPA8 in Parkinson's disease: insights from a Mexican mestizo population.

Parkinson's disease (PD) is a complex neurodegenerative condition characterized by alpha-synuclein aggregation and dysfunctional protein degradation pathways. This study investigates the differential gene expression of pivotal components (UBE2K, PSMC4, SKP1, and HSPA8) within these pathways in a Mexican-Mestizo PD population compared to healthy controls. We enrolled 87 PD patients and 87 controls, assessing their gene expression levels via RT-qPCR. Our results reveal a significant downregulation of PSMC4, SKP1, and HSPA8 in the PD group (p = 0.033, p = 0.003, and p = 0.002, respectively). Logistic regression analyses establish a strong association between PD and reduced expression of PSMC4, SKP1, and HSPA8 (OR = 0.640, 95% CI = 0.415-0.987; OR = 0.000, 95% CI = 0.000-0.075; OR = 0.550, 95% CI = 0.368-0.823, respectively). Conversely, UBE2K exhibited no significant association or expression difference between the groups. Furthermore, we develop a gene expression model based on HSPA8, PSMC4, and SKP1, demonstrating robust discrimination between healthy controls and PD patients. Notably, the model's diagnostic efficacy is particularly pronounced in early-stage PD. In conclusion, our study provides compelling evidence linking decreased gene expression of PSMC4, SKP1, and HSPA8 to PD in the Mexican-Mestizo population. Additionally, our gene expression model exhibits promise as a diagnostic tool, particularly for early-stage PD diagnosis.

EDITOR SPOTLIGHT: Interview with Brain Proteostasis Special Issue Guest Editor Mychael Lourenco.

Mychael Lourenco is an Assistant Professor of Neuroscience at the Institute of Medical Biochemistry Leopoldo de Meis, Federal University of Rio de Janeiro. Research in his lab focusses on understanding the molecular mechanisms underlying cognitive impairment in neurodegeneration and his research on Alzheimer's disease has been recognized by many awards both in Brazil and internationally. He serves as a Reviews Editor for the Journal of Neurochemistry and led this special issue on Brain Proteostasis as a Guest Editor. Here we interviewed him to hear his thoughts on the future of neuroscience and on career development and training.

Autophagy signaling in hypertrophied muscles of diabetic and control rats.

Autophagy plays a vital role in cell homeostasis by eliminating nonfunctional components and promoting cell survival. Here, we examined the levels of autophagy signaling proteins after 7 days of overload hypertrophy in the extensor digitorum longus (EDL) and soleus muscles of control and diabetic rats. We compared control and 3-day streptozotocin-induced diabetic rats, an experimental model for type 1 diabetes mellitus (T1DM). EDL muscles showed increased levels of basal autophagy signaling proteins. The diabetic state did not affect the extent of overload-induced hypertrophy or the levels of autophagy signaling proteins (p-ULK1, Beclin-1, Atg5, Atg12-5, Atg7, Atg3, LC3-I and II, and p62) in either muscle. The p-ULK-1, Beclin-1, and p62 protein expression levels were higher in the EDL muscle than in the soleus before the hypertrophic stimulus. On the contrary, the soleus muscle exhibited increased autophagic signaling after overload-induced hypertrophy, with increases in Beclin-1, Atg5, Atg12-5, Atg7, Atg3, and LC3-I expression in the control and diabetic groups, in addition to p-ULK-1 in the control groups. After hypertrophy, Beclin-1 and Atg5 levels increased in the EDL muscle of both groups, while p-ULK1 and LC3-I increased in the control group. In conclusion, the baseline EDL muscle exhibited higher autophagy than the soleus muscle. Although TDM1 promotes skeletal muscle mass loss and strength reduction, it did not significantly alter the extent of overload-induced hypertrophy and autophagy signaling proteins in EDL and soleus muscles, with the two groups exhibiting different patterns of autophagy activation.

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

Protein degradation-associated mechanisms that are affected in Alzheimer´s disease.

Alzheimer's disease (AD) is the most common type of dementia associated with age-related neurodegeneration. Alteration of several molecular mechanisms has been correlated with the progression of AD. In recent years, dysregulation of proteostasis-associated pathways has emerged as a potential risk factor for neurodegenerative diseases. This review investigated the ubiquitin-proteasome system, lysosome-associated degradation, endoplasmic-reticulum-associated degradation, and the formation of advanced glycation end products. These pathways involved in proteostasis have been reported to be altered in AD, suggesting that their study may be critical for identifying new biomarkers and target molecules for AD.

Use of Heating Methods and Xylose to Increase Rumen Undegradable Protein of Alternative Protein Sources: 1) Peanut Meal.

Peanut meal has an excellent total protein content but also has low rumen undegradable protein (RUP). High-performance ruminants have high RUP requirements. We aimed to evaluate the effects of processing peanut meal with an autoclave and conventional and microwave ovens, with and without using xylose on its ruminal kinetics degradation parameters and intestinal digestibility (ID). In situ studies were conducted to determine dry matter (DM) and crude protein (CP) rumen degradation kinetics. In vitro studies were conducted to evaluate intestinal digestibility (ID). The control treatment had a greater fraction A for DM and CP than peanut meals processed with an autoclave or conventional oven. The control had greater kd for CP compared with the microwave. The addition of xylose decreased fraction A, the degradation rate of fraction B (kd) and RUP, and increased the protein B fraction of autoclaved peanut meal. We observed a decrease in effective degradability (ED) and increased RUP for processed treatments in all experiments compared with the control. Processing methods did not affect the protein ID of autoclaved peanut meal compared to the control. An interaction between xylose and heating time was observed, where increasing heating time linearly reduced the ID of xylose-untreated treatments. Overall, these results suggest that the tested methods effectively increased the RUP content of peanut meal.

Use of Heating Methods and Xylose to Increase Rumen Undegradable Protein of Alternative Protein Sources: 2) Cottonseed Meal.

The ruminal kinetics of protein sources may be changed by heat and sugar treatments. Thus, these processing methods may be used as alternatives to increase beef-cattle diets' rumen undegradable protein (RUP). We aimed to evaluate the effects of processing cottonseed meals with autoclave, conventional, and microwave ovens, with and without using xylose, on the ruminal kinetics degradation parameters and intestinal digestibility (ID). In situ studies were conducted, and each sample was incubated in the rumen to determine dry matter (DM) and crude protein (CP) rumen degradation kinetics. In vitro studies were also conducted to evaluate ID. The control treatment had a greater soluble fraction for DM and CP than processed cottonseed meals (p < 0.05). The addition of xylose decreased both DM and CP water-soluble fractions (fraction A) of cottonseed meal heated in a conventional oven (p < 0.05). Compared to the control, we observed a decrease in effective degradability and increased RUP for all processed methods (p < 0.05). Furthermore, conventional and microwave ovens showed greater ID than the control. Moreover, xylose-treated groups heated in the autoclave and conventional ovens had greater ID than xylose-untreated cottonseed meal. Under these experimental conditions, cottonseed RUP was increased by the evaluated processing methods.

Oomycete intracellular effectors: specialised weapons targeting strategic plant processes.

Oomycete phytopathogens have adapted to colonise plants using effectors as their molecular weapons. Intracellular effectors, mostly proteins but also small ribonucleic acids, are delivered by the pathogens into the host cell cytoplasm where they interfere with normal plant physiology. The diverse host processes emerging as 'victims' of these 'specialised bullets' include gene transcription and RNA-mediated silencing, cell death, protein stability, protein secretion and autophagy. Some effector targets are directly involved in defence execution, while others participate in fundamental metabolisms whose alteration collaterally affects defences. Other effector targets are susceptibility factors (SFs), that is host components that make plants vulnerable to pathogens. SFs are mostly negative regulators of immunity, but some seem necessary to sustain or promote pathogen colonisation.

Leucine-Rich Diet Improved Muscle Function in Cachectic Walker 256 Tumour-Bearing Wistar Rats.

Skeletal muscle atrophy occurs in several pathological conditions, such as cancer, especially during cancer-induced cachexia. This condition is associated with increased morbidity and poor treatment response, decreased quality of life, and increased mortality in cancer patients. A leucine-rich diet could be used as a coadjutant therapy to prevent muscle atrophy in patients suffering from cancer cachexia. Besides muscle atrophy, muscle function loss is even more important to patient quality of life. Therefore, this study aimed to investigate the potential beneficial effects of leucine supplementation on whole-body functional/movement properties, as well as some markers of muscle breakdown and inflammatory status. Adult Wistar rats were randomly distributed into four experimental groups. Two groups were fed with a control diet (18% protein): Control (C) and Walker 256 tumour-bearing (W), and two other groups were fed with a leucine-rich diet (18% protein + 3% leucine): Leucine Control (L) and Leucine Walker 256 tumour-bearing (LW). A functional analysis (walking, behaviour, and strength tests) was performed before and after tumour inoculation. Cachexia parameters such as body weight loss, muscle and fat mass, pro-inflammatory cytokine profile, and molecular and morphological aspects of skeletal muscle were also determined. As expected, Walker 256 tumour growth led to muscle function decline, cachexia manifestation symptoms, muscle fibre cross-section area reduction, and classical muscle protein degradation pathway activation, with upregulation of FoxO1, MuRF-1, and 20S proteins. On the other hand, despite having no effect on the walking test, inflammation status or muscle oxidative capacity, the leucine-rich diet improved muscle strength and behaviour performance, maintained body weight, fat and muscle mass and decreased some protein degradation markers in Walker 256 tumour-bearing rats. Indeed, a leucine-rich diet alone could not completely revert cachexia but could potentially diminish muscle protein degradation, leading to better muscle functional performance in cancer cachexia.

Sleep debt induces skeletal muscle injuries in athletes: A promising hypothesis.

Sleep is a physiological state and it is fundamental for physical and cognitive recovery of athletes. Due to strenuous training and competitions, athletes may present sleep complaints compromising good quality and quantity of sleep. Studies have related sleep debt to the occurrence of musculoskeletal injuries in athletes, but the mechanisms that can lead to this are not entirely clear. Studies involving animals and humans have shown that poor sleep quality can cause significant changes in hormones and cytokines. Demonstrating that this hormones changes lead to a decrease of testosterone and growth hormone levels and increased cortisol levels, important hormones in the process of protein synthesis and degradation. In athletes, the sport itself is a risk factor of injuries, and sleep debt may result in overtraining syndrome associated with inflammatory markers and ultimately to immune system dysfunction. Thus, we hypothesize that athletes who have sleep debt are more susceptible to musculoskeletal injuries due to increased catabolic pathway signaling, i.e. protein degradation and decreased anabolic pathway signaling, compromising muscle integrity. In this sense, we indicate the relationship between musculoskeletal injuries and sleep debt involving new targets for immunological signaling pathways that start the reduction of the muscle recovery process.

The yeast protein Ubx4p contributes to mitochondrial respiration and lithium-galactose-mediated activation of the unfolded protein response.

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

The biology of the adenovirus E1B 55K protein.

The adenovirus E1B 55K (E1B) protein plays major roles in productive adenoviral infection and cellular transformation. Interest in E1B increased because of the potential of adenoviruses as therapeutic vectors, and the E1B gene is commonly deleted from adenovirus vectors for anticancer therapy. E1B activities are spatiotemporally regulated through SUMOylation and phosphorylation, and through interactions with multiple partners that occur presumably at different intracellular sites and times postinfection. E1B is implicated in the formation of viral replication compartments and regulates viral genome replication and transcription, transcriptional repression, degradation of cellular proteins, and several intranuclear steps of viral late mRNA biogenesis. Here, we review advances in our understanding of E1B during productive adenovirus replication and discuss fundamental aspects that remain unresolved.

Chloroplast Protein Degradation in Senescing Leaves: Proteases and Lytic Compartments.

Leaf senescence is characterized by massive degradation of chloroplast proteins, yet the protease(s) involved is(are) not completely known. Increased expression and/or activities of serine, cysteine, aspartic, and metalloproteases were detected in senescing leaves, but these studies have not provided information on the identities of the proteases responsible for chloroplast protein breakdown. Silencing some senescence-associated proteases has delayed progression of senescence symptoms, yet it is still unclear if these proteases are directly involved in chloroplast protein breakdown. At least four cellular pathways involved in the traffic of chloroplast proteins for degradation outside the chloroplast have been described (i.e., "Rubisco-containing bodies," "senescence-associated vacuoles," "ATI1-plastid associated bodies," and "CV-containing vesicles"), which differ in their dependence on the autophagic machinery, and the identity of the proteins transported and/or degraded. Finding out the proteases involved in, for example, the degradation of Rubisco, may require piling up mutations in several senescence-associated proteases. Alternatively, targeting a proteinaceous protein inhibitor to chloroplasts may allow the inhibitor to reach "Rubisco-containing bodies," "senescence-associated vacuoles," "ATI1-plastid associated bodies," and "CV-containing vesicles" in essentially the way as chloroplast-targeted fluorescent proteins re-localize to these vesicular structures. This might help to reduce proteolytic activity, thereby reducing or slowing down plastid protein degradation during senescence.

Nuclear proteasomal degradation of Saccharomyces cerevisiae inorganic pyrophosphatase Ipp1p, a nucleocytoplasmic protein whose stability depends on its subcellular localization.

Inorganic pyrophosphate (PPi) is an abundant by-product of cellular metabolism. PPi-producing reactions take place in the nucleus concurrently with reactions that use PPi as a substrate. Saccharomyces cerevisiae possesses two soluble pyrophosphatases (sPPases): Ipp1p, an essential and allegedly cytosolic protein, and Ipp2p, a mitochondrial isoenzyme. However, no sPPase has yet been unambiguously described in the nucleus. In vivo studies with fluorescent fusions together with activity and immunodetection analyses demonstrated that Ipp1p is a nucleocytoplasmic protein. Mutagenesis analysis showed that this sPPase possesses a nuclear localization signal which participates in its nuclear targeting. Enforced nucleocytoplasmic targeting by fusion to heterologous nuclear import and export signals caused changes in polypeptide abundance and activity levels, indicating that Ipp1p is less stable in the nucleus that in the cytoplasm. Low nuclear levels of this sPPase are physiologically relevant and may be related to its catalytic activity, since cells expressing a functional nuclear-targeted chimaera showed impaired growth and reduced chronological lifespan, while a nuclear-targeted catalytically inactive protein was not degraded and accumulated in the nucleus. Moreover, nuclear proteasome inhibition stabilized Ipp1p whereas nuclear targeting promoted its ubiquitination and interaction with Ubp3p, a component of the ubiquitin-proteasome system. Overall, our results indicate that Ipp1p is nucleocytoplasmic, that its stability depends on its subcellular localization and that sPPase catalytic competence drives its nuclear degradation through the ubiquitin-proteasome system. This suggests a new scenario for PPi homeostasis where both nucleocytoplasmic transport and nuclear proteasome degradation of the sPPase should contribute to control nuclear levels of this ubiquitous metabolite.

Short-term creatine supplementation changes protein metabolism signaling in hindlimb suspension

The effect of a short-term creatine supplementation on hindlimb suspension (HS)-induced muscle atrophy was investigated. Creatine monohydrate (5 g/kg b.w. per day) or placebo, divided in 2 daily doses, was given by oral gavage for 5 days. Rats were maintained in HS with dietary supplementation concomitantly for 5 days. Body weight, soleus and EDL muscle masses, and cross-sectional areas (CSA) of the muscle fibers were measured. Signaling pathways associated with skeletal muscle mass regulation (FST, MSTN, FAK, IGF-1, MGF, Akt, mTOR, atrogin-1, and MuRF1 expressions, and Akt, S6, GSK3B, and 4EBP1 proteins) were evaluated in the muscles. Soleus muscle exhibited more atrophy than the EDL muscle due to HS. Creatine supplementation attenuated the decrease of wet weight and increased p-4EBP1 protein in the EDL muscle of HS rats. Also, creatine increased mTOR and atrogin-1 expressions in the same muscle and condition. In the absence of HS, creatine supplementation increased FAK and decreased MGF expressions in the EDL muscle. Creatine attenuated the increase in FST expression due to HS in the soleus muscle. MuRF1 expression increased in the soleus muscle due to creatine supplementation in HS animals whereas atrogin-1 expression increased still further in this group compared with untreated HS rats. In conclusion, short-term creatine supplementation changed protein metabolism signaling in soleus and EDL muscles. However, creatine supplementation only slightly attenuated the mass loss of both muscles and did not prevent the CSA reduction and muscle strength decrease induced by HS for 5 days.

Calcium sensing receptor activates the NLRP3 inflammasome via a chaperone-assisted degradative pathway involving Hsp70 and LC3-II.

Calcium sensing receptor (CaSR) activates the NLRP3 inflammasome with consequences on homeostatic responses. However, little is known about how this process is orchestrated. Since proteolysis of critical regulators of NLRP3 inflammasome contribute to its activation, we aimed to understand how CaSR stimulates proteolytic pathways to activate the NLRP3 inflammasome. We found that proteasome and lysosome-dependent mechanisms are activated by CaSR to promote the degradation of important regulators of NLRP inflammasome. The pathway involves Gαq/PLC/PKC and Gßγ/PI3K signaling cascades and IRAK1 ubiquitination. In addition, CaSR stimulates Hsp70 expression activating a chaperone-assisted protein degradation that dictates the fate of ASC, NLRP3 (NOD-like receptor family protein 3), IRAK1 and TRAF6 proteins, turning on the NLRP3 inflammasome. In response to CaSR signaling, these proteins are degraded through the combination of CUPS (chaperone-assisted ubiquitin proteasome pathway) and CAEMI (chaperone-assisted endosomal microautophagy) systems being integrated by autophagosomes (chaperone-assisted macroautophagy, CAMA), as indicated by LC3-II, a classical marker for autophagy, that is induced in the process. Furthermore, CaSR triggers the proteolytic cleavage of pro-IL-1ß (IL-1ß, 31 kDa) into mature IL-1ß (IL-1ß, 17 kDa), via the proteasome. Taken together, our results indicate that CaSR promotes NLRP3 inflammasome activation and proteolytic maturation of IL-1ß by inducing CUPS and CAEMI, chaperone-assisted degradation pathways. Overall, these results support the inclusion of CaSR as an activator of homeostasis-altering molecular processes.

Effluent composition prediction of a two-stage anaerobic digestion process: machine learning and stoichiometry techniques.

Computational self-adapting methods (Support Vector Machines, SVM) are compared with an analytical method in effluent composition prediction of a two-stage anaerobic digestion (AD) process. Experimental data for the AD of poultry manure were used. The analytical method considers the protein as the only source of ammonia production in AD after degradation. Total ammonia nitrogen (TAN), total solids (TS), chemical oxygen demand (COD), and total volatile solids (TVS) were measured in the influent and effluent of the process. The TAN concentration in the effluent was predicted, this being the most inhibiting and polluting compound in AD. Despite the limited data available, the SVM-based model outperformed the analytical method for the TAN prediction, achieving a relative average error of 15.2% against 43% for the analytical method. Moreover, SVM showed higher prediction accuracy in comparison with Artificial Neural Networks. This result reveals the future promise of SVM for prediction in non-linear and dynamic AD processes. Graphical abstract ᅟ.