Proteolytic Complex Research Articles

Beneficial Effects Induced by a Proprietary Blend of a New Bromelain-Based Polyenzymatic Complex Plus N-Acetylcysteine in Urinary Tract Infections: Results from In Vitro and Ex Vivo Studies.

Background/Objectives: Urinary tract infections (UTIs) are infections that involve the urethra, bladder, and, in much more severe cases, even kidneys. These infections represent one of the most common diseases worldwide. Various pathogens are responsible for this condition, the most common being Escherichia coli (E. coli). Bromelain is a proteolytic complex obtained from the stem and stalk of Ananas comosus (L.) Merr. showing several beneficial activities. In addition to bromelain, N-acetylcysteine (NAC) has also been used. Methods: The purpose of this experiment was to evaluate the antibacterial, anti-motility, and anti-biofilm effects of a new polyenzymatic complex (DIF17BRO®) in combination with NAC (the Formulation) on various strains of E. coli isolated from patients with UTIs. Subsequently, the anti-inflammatory and antioxidant effects of the Formulation were studied in an ex vivo model of cystitis, using bladder samples from mice exposed to E. coli lipopolysaccharide (LPS). Results: Our results showed that the Formulation significantly affects the capability of bacteria to form biofilm and reduces the bacteria amount in the mature biofilm. Moreover, it combines the interesting properties of NAC and a polyenzyme plant complex based on bromelain in a right dose to affect the E. coli adhesion capability. Finally, the Formulation exhibited protective effects, as confirmed by the inhibitory activities on multiple inflammatory and oxidative stress-related pathways on bladder specimens exposed to LPS. Conclusions: This blend of active compounds could represent a promising and versatile approach to use to overcome the limitations associated with conventional therapies.

Structural insights into the membrane-bound proteolytic machinery of bacterial protein quality control.

In bacteria and eukaryotic organelles of prokaryotic origin, ATP-dependent proteases are crucial for regulating protein quality control through substrate unfolding and degradation. Understanding the mechanism and regulation of this key cellular process could prove instrumental in developing therapeutic strategies. Very recently, cryo-electron microscopy structural studies have shed light on the functioning of AAA+ proteases, including membrane-bound proteolytic complexes. This review summarizes the structure and function relationship of bacterial AAA+ proteases, with a special focus on the sole membrane-bound AAA+ protease in Escherichia coli, FtsH. FtsH substrates include both soluble cytoplasmic and membrane-incorporated proteins, highlighting its intricate substrate recognition and processing mechanisms. Notably, 12 copies of regulatory HflK and HflC proteins, arranged in a cage-like structure embedded in the bacterial inner membrane, can encase up to 4 FtsH hexamers, thereby regulating their role in membrane protein quality control. FtsH represents an intriguing example, highlighting both its similarity to cytosolic AAA+ proteases with respect to overall architecture and oligomerization as well as its unique features, foremost its incorporation into a membrane-bound complex formed by HflK and HflC to mediate its function in protein quality control.

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB’s low specificity for Tv20S compared to the human proteasome and demonstrate CP-17’s higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

Inhibition of nucleo-cytoplasmic proteasome translocation by the aromatic amino acids or silencing Sestrin3-their sensing mediator-is tumor suppressive.

The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response ismediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further showthat the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer.

Proteolytic Regulation in the Biosynthesis of Natural Product Via a ClpP Protease System.

Protein degradation is a tightly regulated biological process that maintains bacterial proteostasis. ClpPs are a highly conserved family of serine proteases that associate with the AAA + ATPase (an ATPase associated with diverse cellular activities) to degrade protein substrates. Identification and biochemical characterization of protein substrates for the AAA + ATPase-dependent ClpP degradation systems are considered essential for gaining an understanding of the molecular operation of the complex ClpP degradation machinery. Consequently, expanding the repertoire of protein substrates that can be degraded in vitro and within bacterial cells is necessary. Here, we report that AAA + ATPase-ClpP proteolytic complexes promote degradation of the secondary metabolite surfactin synthetases SrfAA, SrfAB, and SrfAC in Bacillus subtilis. On the basis of in vitro and in-cell studies coupled with activity-based protein profiling of nonribosomal peptide synthetases, we showed that SrfAC is targeted to the ClpC-ClpP proteolytic complex, whereas SrfAA is hydrolyzed not only by the ClpC-ClpP proteolytic complex but also by different ClpP proteolytic complexes. Furthermore, SrfAB does not appear to be a substrate for the ClpC-ClpP proteolytic complex, thereby implying that other ClpP proteolytic complexes are involved in the degradation of this surfactin synthetase. Natural product biosynthesis is regulated by the AAA + ATPase-ClpP degradation system, indicating that protein degradation plays a role in the regulatory stages of biosynthesis. However, few studies have examined the regulation of protein degradation levels. Furthermore, SrfAA, SrfAB, and SrfAC were identified as protein substrates for AAA + ATPase-ClpP degradation systems, thereby contributing to a better understanding of the complex ClpP degradation machinery.

First Insight into the Degradome of Aspergillus ochraceus: Novel Secreted Peptidases and Their Inhibitors.

Aspergillus fungi constitute a pivotal element within ecosystems, serving as both contributors of biologically active compounds and harboring the potential to cause various diseases across living organisms. The organism's proteolytic enzyme complex, termed the degradome, acts as an intermediary in its dynamic interaction with the surrounding environment. Using techniques such as genome and transcriptome sequencing, alongside protein prediction methodologies, we identified putative extracellular peptidases within Aspergillus ochraceus VKM-F4104D. Following manual annotation procedures, a total of 11 aspartic, 2 cysteine, 2 glutamic, 21 serine, 1 threonine, and 21 metallopeptidases were attributed to the extracellular degradome of A. ochraceus VKM-F4104D. Among them are enzymes with promising applications in biotechnology, potential targets and agents for antifungal therapy, and microbial antagonism factors. Thus, additional functionalities of the extracellular degradome, extending beyond mere protein substrate digestion for nutritional purposes, were demonstrated.

Digestion inhibition of potato starch-botanical proteolytic polypeptides complexes subjected to heat moisture treatment based on steric hindrance and enzymatic inhibition

The effects of food components on starch digestibility is a prominent topic within the realms of food and nutritional science. In this study, complexes of potato starch (PS) and botanical proteolytic polypeptides (BPPs), including wheat peptides, maize peptides, rice peptides, and soybean peptides, were subjected to heat-moisture treatment (HMT). The physicochemical, structural, and digestibility variations of the PS-BPPs complexes were analyzed using polarized light microscopy, DSC, X-ray, FT-IR, SAXS, and in vitro simulated digestion. Additionally, we evaluated the inhibitory effects of BPPs on porcine pancreatic α-amylase (PPA). The results showed that the polarization cross of PS-BPPs complexes was weakened after HMT. The gelatinization temperatures (To, Tp, Tc) of the PS-BPPs complexes increased significantly. The starch in the PS-BPPs complexes transitioned from B-type structure to A-type structure after HMT, accompanied by an increase in short-range ordering and semicrystalline lamella thickness. In particular, the complexes exhibited higher contents of slowly digestible starch (SDS) and resistant starch (RS). Furthermore, wheat, rice, and soybean peptides exhibited a mixed type of competitive inhibition against PPA, whereas maize peptides exhibited competitive inhibition. The findings provide valuable insights into the role of BPPs in regulating starch digestion. Moreover, BPPs hold potential for enhancing the nutritional value and health benefits of foods.

Mechanism of complement inhibition by a mosquito protein revealed through cryo-EM

Salivary complement inhibitors occur in many of the blood feeding arthropod species responsible for transmission of pathogens. During feeding, these inhibitors prevent the production of proinflammatory anaphylatoxins, which may interfere with feeding, and limit formation of the membrane attack complex which could damage arthropod gut tissues. Salivary inhibitors are, in many cases, novel proteins which may be pharmaceutically useful or display unusual mechanisms that could be exploited pharmaceutically. Albicin is a potent inhibitor of the alternative pathway of complement from the saliva of the malaria transmitting mosquito, Anopheles albimanus. Here we describe the cryo-EM structure of albicin bound to C3bBb, the alternative C3 convertase, a proteolytic complex that is responsible for cleavage of C3 and amplification of the complement response. Albicin is shown to induce dimerization of C3bBb, in a manner similar to the bacterial inhibitor SCIN, to form an inactive complex unable to bind the substrate C3. Size exclusion chromatography and structures determined after 30 minutes of incubation of C3b, factor B (FB), factor D (FD) and albicin indicate that FBb dissociates from the inhibited dimeric complex leaving a C3b-albicin dimeric complex which apparently decays more slowly.

New Method of Obtaining Proteolytic Enzymatic Preparation from Fungal Strain Fusarium gibbosum CNMN FD 12

Proteases, that are able to hydrolyze the peptide bonds in proteins and polypeptides, occupy a key position in diverse fields, such as food, agriculture, animal feed. Considering the increasing demand for microbial proteases, identification of new way for improving the productivity of producers is of great importance. The aim of the present study was to evaluate the effect of a new coordination compound [Fe(H2L)(H2O)2](NO3)3∙2.5H2O (where H2L = 2,6-diacetylpyridine bis(picolinoylhydrazone)) on the activity of proteolytic complex synthesized by the micromycete Fusarium gibbosum CNMN FD 12 under the submerged fermentations in shake flask, as well as in lab-scale fermenter. It was found that [Fe(H2L)(H2O)2](NO3)3∙2.5H2O exerts a significant positive effect on proteolytic activity in F. gibbosum CNMN FD 12 both in flasks and bioreactor and can be used as stimulator and regulator of proteases activity. By varying the concentration of the coordination compound and the duration of cultivation, it is possible to obtain proteolytic complexes enriched in different types of proteases (acid, neutral or alkaline), depending on the practical needs.

Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

IRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

Interactome Analysis of ClpX Reveals Its Regulatory Role in Metabolism and Photosynthesis in Cyanobacteria.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

Decreased Proteasomal Function Exacerbates Endoplasmic Reticulum Stress-Induced Chronic Inflammation in Obese Adipose Tissue

Low-grade chronic inflammation contributes to both aging and the pathogenesis of age-related diseases. White adipose tissue (WAT) in obese individuals exhibits chronic inflammation, which is associated with obesity-related disorders. Aging exacerbates obesity-related inflammation in WAT; however, the molecular mechanisms underlying chronic inflammation and its exacerbation by aging remain unclear. Age-related decline in activity of the proteasome, a multi-subunit proteolytic complex, has been implicated in age-related diseases. This study employed a mouse model with decreased proteasomal function that exhibits age-related phenotypes to investigate the impact of adipocyte senescence on WAT inflammation. Transgenic mice expressing proteasomal subunit β5t with weak chymotrypsin-like activity suffer from reduced lifespan and develop age-related phenotypes. Mice fed with a high-fat diet (HFD) and suffering proteasomal dysfunction exhibited increased WAT inflammation, increased infiltration of pro-inflammatory M1-like macrophages, and increased pro-inflammatory adipocytokines like monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and tumor necrosis factor αwhich are all associated with activation of endoplasmic reticulum (ER) stress-related pathways. Impaired proteasomal activity also activated ER stress-related molecules and induced expression of pro-inflammatory adipocytokines in adipocyte-like cells differentiated from 3T3-L1 cells. Collective evidence suggests that impaired proteasomal activity increases ER stress and that subsequent inflammatory pathways play pivotal roles in WAT inflammation. Since proteasomal function declines with age, age-related proteasome impairment may be involved in obesity-related inflammation among the elderly.

CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.

Diverse nature of ClpX degradation motifs in Streptococcus mutans.

Cytoplasmic Clp-related proteases play a major role in maintaining cellular proteome in bacteria. ClpX/P is one such proteolytic complex that is important for conserving protein homeostasis. In this study, we investigated the role of ClpX/P in Streptococcus mutans, an important oral pathogen. We identified several putative substrates whose cellular levels are regulated by ClpX/P in S. mutans and subsequently discovered several recognition motifs that are critical for degradation. Our study is the first comprehensive analysis of determining ClpX/P motifs in streptococci. We believe that identifying the substrates that are regulated by ClpX/P will enhance our understanding about virulence regulation in this important group of pathogens.

NlpI-Prc Proteolytic Complex Mediates Peptidoglycan Synthesis and Degradation via Regulation of Hydrolases and Synthases in Escherichia coli.

Balancing peptidoglycan (PG) synthesis and degradation with precision is essential for bacterial growth, yet our comprehension of this intricate process remains limited. The NlpI-Prc proteolytic complex plays a crucial but poorly understood role in the regulation of multiple enzymes involved in PG metabolism. In this paper, through fluorescent D-amino acid 7-hydroxycoumarincarbonylamino-D-alanine (HADA) labeling and immunolabeling assays, we have demonstrated that the NlpI-Prc complex regulates the activity of PG transpeptidases and subcellular localization of PBP3 under certain growth conditions. PBP7 (a PG hydrolase) and MltD (a lytic transglycosylase) were confirmed to be negatively regulated by the NlpI-Prc complex by an in vivo degradation assay. The endopeptidases, MepS, MepM, and MepH, have consistently been demonstrated as redundantly essential "space makers" for nascent PG insertion. However, we observed that the absence of NlpI-Prc complex can alleviate the lethality of the mepS mepM mepH mutant. A function of PG lytic transglycosylases MltA and MltD as "space makers" was proposed through multiple gene deletions. These findings unveil novel roles for NlpI-Prc in the regulation of both PG synthesis and degradation, shedding light on the previously undiscovered function of lytic transglycosylases as "space makers" in PG expansion.

An Immunoproteasome Activator That Increases MHC Class I Antigen Presentation to Enhance Anti-Tumor Immunity

Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Proteasomes are multi-subunit proteolytic complexes that selectively degrade intracellular proteins. Proteasomes play a critical in controlling myriad cellular processes including metabolism, cell cycle progression, signal transduction, protein quality control and differentiation. Importantly, proteasomes are also the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8 and 9-amino acid peptides, which are then transported into the endoplasmic reticulum, where they associate with MHC-class I molecules and are presented to the cell surface. Immune cells contain a highly specialized, cytokine-inducible proteasome variant, known as the immunoproteasome, in which the three constitutive catalytic subunits are replaced by homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8 + T-cells. Here, we performed a high-through screen (HTS) to detect novel molecular entities that activated immuno-proteasomal catalytic activity. We identified a curated panel of molecules which not only enhanced hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin ( OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by murine MHC-class I H-2Kb molecules. E.G7-Ova cells were pretreated with compound A and co-cultured with the T-cell hybridoma B3Z that had been genetically-engineered to express a SIINFEKL-restricted t-cell receptor (TCR). Pretreatment of E.G7-Ova cells with compound A significantly increased tumor cell lysis by B3Z cells. The treatment of multiple myeloma (MM) cells with compound A also dramatically enhanced the expression of pan-MHC class I antigens. Treatment of MM cells with compound A increased the intracellular level of the proteasome activator PA28α/β, which is expressed by PSME1/2. Compound A treatment also enhanced hydrolysis of the fluorogenic peptide substrate Ac-ANW-AMC, which is selectively cleaved by the immunoproteasome β5-associated catalytic activity. Treatment of MM cells with compound A followed by mass spectrometry (MS) revealed that immunoproteasome activation modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules. Pre-treatment of MM cells with compound A also significantly enhanced the anti-myeloma activity of human cytotoxic T-cells. Taken together, our results demonstrate that immunoproteasome activation has the potential to overcome immune escape and promote host immunity. In addition, our model systems now allows us to identify actionable tumor antigens on patient tumor cells as an indispensable tool for the development of cancer immunotherapies, e.g., T cell receptor (TCR)-transduced T-cells and patient-specific mRNA and peptide vaccines. Finally, immunoproteasome activators have the potential to contribute not only to cytotoxic T-cell mediated tumor lysis but also the treatment of intracellular infections and autoimmune and neurodegenerative proteinopathies characterized by the pathologic accumulation of toxic, proteinaceous aggregates.

Comparative genomics of the proteostasis network in extreme acidophiles.

Extreme acidophiles thrive in harsh environments characterized by acidic pH, high concentrations of dissolved metals and high osmolarity. Most of these microorganisms are chemolithoautotrophs that obtain energy from low redox potential sources, such as the oxidation of ferrous ions. Under these conditions, the mechanisms that maintain homeostasis of proteins (proteostasis), as the main organic components of the cells, are of utmost importance. Thus, the analysis of protein chaperones is critical for understanding how these organisms deal with proteostasis under such environmental conditions. In this work, using a bioinformatics approach, we performed a comparative genomic analysis of the genes encoding classical, periplasmic and stress chaperones, and the protease systems. The analysis included 35 genomes from iron- or sulfur-oxidizing autotrophic, heterotrophic, and mixotrophic acidophilic bacteria. The results showed that classical ATP-dependent chaperones, mostly folding chaperones, are widely distributed, although they are sub-represented in some groups. Acidophilic bacteria showed redundancy of genes coding for the ATP-independent holdase chaperones RidA and Hsp20. In addition, a systematically high redundancy of genes encoding periplasmic chaperones like HtrA and YidC was also detected. In the same way, the proteolytic ATPase complexes ClpPX and Lon presented redundancy and broad distribution. The presence of genes that encoded protein variants was noticeable. In addition, genes for chaperones and protease systems were clustered within the genomes, suggesting common regulation of these activities. Finally, some genes were differentially distributed between bacteria as a function of the autotrophic or heterotrophic character of their metabolism. These results suggest that acidophiles possess an abundant and flexible proteostasis network that protects proteins in organisms living in energy-limiting and extreme environmental conditions. Therefore, our results provide a means for understanding the diversity and significance of proteostasis mechanisms in extreme acidophilic bacteria.

Gene identification and RNAi-silencing of p62/SQSTM1 in the vector Rhodnius prolixus reveals a high degree of sequence conservation but no apparent deficiency-related phenotypes in vitellogenic females.

Autophagy and the ubiquitin-proteasome system (UPS) are important cellular mechanisms that coordinate protein degradation essential for proteostasis. P62/SQSTM1 is a receptor cargo protein able to deliver ubiquitinated targets to the proteasome proteolytic complex and/or to the autophagosome. In the insect vector of Chagas disease, Rhodnius prolixus, previous works have shown that the knockdown of different autophagy-related genes (ATGs) and ubiquitin-conjugating enzymes resulted in abnormal oogenesis phenotypes and embryo lethality. Here, we investigate the role of the autophagy/UPS adaptor protein p62 during the oogenesis and reproduction of this vector. We found that R. prolixus presents one isoform of p62 encoded by a non-annotated gene. The predicted protein presents the domain architecture anticipated for p62: PB1 (N-term), ZZ-finger, and UBA (C-term) domains, and phylogenetic analysis showed that this pattern is highly conserved within insects. Using parental RNAi, we found that although p62 is expressed in the ovary, midgut, and fat body of adult females, systemic silencing of this gene did not result in any apparent phenotypes under in-house conditions. The insects' overall levels of blood meal digestion, lifespan, yolk protein production, oviposition, and embryo viability were not altered when compared to controls. Because it is known that autophagy and UPS can undergo compensatory mechanisms, we asked whether the silencing of p62 was triggering adaptative changes in the expression of genes of the autophagy, UPS, and the unfolded protein response (UPR) and found that only ATG1 was slightly up regulated in the ovaries of silenced females. In addition, experiments to further investigate the role of p62 in insects previously silenced for the E1-conjugating enzyme (a condition known to trigger the upregulation of p62), also did not result in any apparent phenotypes in vitellogenic females.

The Ubiquitin-26S Proteasome Pathway and Its Role in the Ripening of Fleshy Fruits.

The 26S proteasome is an ATP-dependent proteolytic complex in eukaryotes, which is mainly responsible for the degradation of damaged and misfolded proteins and some regulatory proteins in cells, and it is essential to maintain the balance of protein levels in the cell. The ubiquitin-26S proteasome pathway, which targets a wide range of protein substrates in plants, is an important post-translational regulatory mechanism involved in various stages of plant growth and development and in the maturation process of fleshy fruits. Fleshy fruit ripening is a complex biological process, which is the sum of a series of physiological and biochemical reactions, including the biosynthesis and signal transduction of ripening related hormones, pigment metabolism, fruit texture changes and the formation of nutritional quality. This paper reviews the structure of the 26S proteasome and the mechanism of the ubiquitin-26S proteasome pathway, and it summarizes the function of this pathway in the ripening process of fleshy fruits.