Unidentified Protein Research Articles

Proteomic analysis of heat-stable proteins revealed an increased proportion of proteins with compositionally biased regions

Intrinsically disordered proteins (IDPs) have been in the spotlight for their unique properties, such as their lack of secondary structures and low sequence complexity. Alpha-synuclein and tau are representative disease-related IDPs with low complexity regions in their sequences, accumulating in the brains of patients with Parkinson disease and Alzheimer disease, respectively. Their heat resistance in particular was what attracted our attention. We assumed that there exist many other unidentified proteins that are resistant to heat-treatment, referred to as heat-stable proteins, which would also have low sequence complexity. In this study, we performed proteomic analysis of heat-stable proteins of mouse brains and found that proteins with compositionally biased regions are abundant in the heat-stable proteins. The proteins related to neurodegeneration are known to undergo different types of post-translational modifications (PTMs) such as phosphorylation and ubiquitination. We then investigated the heat-stability and aggregation properties of phosphorylated synuclein and tau with different phosphorylation sites. We suggest that PTMs can be important factors that determine the heat-stability and aggregation properties of a protein. IDPs identified in the heat-stable proteins of mouse brains would be candidates for the pathogenic proteins for neurodegeneration.

A new mass spectral library for high-coverage and reproducible analysis of the Plasmodium falciparum-infected red blood cell proteome.

Background Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry–based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility.ResultsHere, we present a comprehensive P. falciparum–infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites.ConclusionA reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

Dual-function Spot 42 RNA encodes a 15–amino acid protein that regulates the CRP transcription factor

SignificanceDual-function RNAs base pair with target messenger RNAs as small regulatory RNAs and encode small protein regulators. However, only a limited number of these dual-function regulators have been identified. In this study, we show that a well-characterized base-pairing small RNA surprisingly also encodes a 15-amino acid protein. The very small protein binds the cyclic adenosine monophosphate receptor protein transcription factor to block activation of some promoters, raising the question of how many other transcription factors are modulated by unidentified small proteins.

Titer improvement of milbemycins via coordinating metabolic competition and transcriptional co-activation controlled by Streptomyces antibiotic regulatory protein family regulator in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is a promising producer of milbemycins (MILs), the macrolide pesticide used widely in agriculture. The relationship between different biosynthetic gene clusters (BGCs) and the MIL BGC remains unclear, which hinders the precise metabolic engineering of S. bingchenggensis for titer improvement. To address this issue, this study discovered the regulatory function of a previously unidentified regulator KelR on a type-II polyketide BGC, MIL BGC, and two other BGCs, and caused titer improvement. First, a type II polyketide synthase (PKS) gene cluster kel with a bidirectional effect on MIL biosynthesis was found using transcriptome analysis. A Streptomyces antibiotic regulatory protein (SARP) family regulator KelR from the kel cluster was then characterized as an activator of several BGCs including mil and kel clusters. Metabolic competition between mil and kel clusters at the late fermentation stage was confirmed. Finally, KelR and those BGCs were manipulated in S. bingchenggensis, which led to a 71.7% titer improvement of MIL A3/A4 to 4058.2 ± 71.0 mg/L. This study deciphered the regulatory function of a previously unidentified regulatory protein KelR on several BGCs including mil in S. bingchenggensis and provided an example of coordinating metabolic competition and coregulation for titer improvement of secondary metabolites.

Identification of multiple isoforms of glucocorticoid receptor in nasal polyps of patients with chronic rhinosinusitis

BackgroundThe conventional belief that glucocorticosteroid (GC) acts through a single brand glucocorticoid receptor (GR)α protein has changed dramatically with the discovery of multiple GR isoforms. We aimed to evaluate whether multiple GR protein isoforms are expressed in chronic rhinosinusitis with nasal polyps (CRSwNP) and whether GR protein isoform expression profiles differ between different endotypes of CRSwNP.MethodsThirty-eight patients with CRSwNP and ten healthy volunteers were included. The protein expression of multiple GR isoforms in nasal polyps (NPs) tissue and control mucosae was examined by western blot analysis with different GR antibodies.ResultsFive bands, including three bands for known proteins (GRα-A/B, GRα-C, and GRα-D) and two bands for unidentified proteins at 67 kilodaltons (kDa) and 60 kDa, were identified with both total GR antibody (PA1-511A) and GRα-specific antibody (PA1-516). GRα-D intensity, which was abundant in nasal mucosa, was significantly increased in the CRSwNP group and was especially elevated in the noneosinophilic CRSwNP (NE-CRSwNP) group (PA1-511A: P < 0.001 and P = 0.0018; PA1-516: P < 0.003 and P = 0.006, respectively). Additionally, the intensities of the newly recognized 67 kDa and 60 kDa bands were much greater in the NE-CRSwNP subgroup than in the eosinophilic CRSwNP (E-CRSwNP) subgroup; in the E-CRSwNP subgroup, the median intensities were even lower than those in the control group.ConclusionsThis study provides evidence that nasal tissues express multiple GR protein isoforms. GR protein isoforms presented disease and tissue-specific expression profiles that differed between the CRSwNP and control groups and between the E-CRSwNP and NE-CRSwNP subgroups.Graphical abstract

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H2O2) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H2O2 in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H2O2 is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H2O2 between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H2O2 permeation. The permeability coefficient of H2O2 through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10−4 and 5 × 10−3 cm s−1, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H2O2 at 37 °C, on the other hand, was 1.6 × 10−3 cm s−1. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H2O2. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H2O2 as normal human RBCs. Therefore, these results indicate that H2O2 does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.

Closing the loop on female fertility.

Discovery of a previously unidentified pituitary protein could provide innovative therapeutic options to regulate female fertility.

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing.

Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2.

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.

Expanding Asgard members in the domain of Archaea sheds new light on the origin of eukaryotes.

The hypothesis that eukaryotes originated from within the domain Archaea has been strongly supported by recent phylogenomic analyses placing Heimdallarchaeota-Wukongarchaeota branch from the Asgard superphylum as the closest known archaeal sister-group to eukaryotes. However, our understanding is still limited in terms of the relationship between eukaryotes and archaea, as well as the evolution and ecological functions of the Asgard archaea. Here, we describe three previously unknown phylum-level Asgard archaeal lineages, tentatively named Sigyn-, Freyr- and Njordarchaeota. Additional members in Wukongarchaeota and Baldrarchaeota from distinct environments are also reported here, further expanding their ecological roles and metabolic capacities. Comprehensive phylogenomic analyses further supported the origin of eukaryotes within Asgard archaea and a new lineage Njordarchaeota was supposed as the known closest branch with the eukaryotic nuclear host lineage. Metabolic reconstruction suggests that Njordarchaeota may have a heterotrophic lifestyle with capability of peptides and amino acids utilization, while Sigynarchaeota and Freyrarchaeota also have the potentials to fix inorganic carbon via the Wood-Ljungdahl pathway and degrade organic matters. Additionally, the Ack/Pta pathway for homoacetogenesis and de novo anaerobic cobalamin biosynthesis pathway were found in Freyrarchaeota and Wukongrarchaeota, respectively. Some previously unidentified eukaryotic signature proteins for intracellular membrane trafficking system, and the homologue of mu/sigma subunit of adaptor protein complex, were identified in Freyrarchaeota. This study expands the Asgard superphylum, sheds new light on the evolution of eukaryotes and improves our understanding of ecological functions of the Asgard archaea.

Galectin 3 Protein Network Analysis By Reverse Protein Arrays Demonstrates Regulation of MCL-1, PKC Delta and CD74 Associated with Poor Survival in Patients with AML

Galectin 3 (LGALS3) is a glycan binding protein that positively regulates survival signaling by diverse mechanisms. LGALS3 is involved in trafficking of kinases such as RAS and receptors such as CD44 to membranes. LGALS3 gene expression is associated with poor survival in AML (Cheng et al Blood 2013) but its functional role is not clear. We therefore analyzed protein expression of LGALS3 and 230 other proteins by RPPA in 511 AML patients. LGALS3 was significantly elevated in AML blast cells compared to normal CD34+ counterparts and highest in monocytic AML patients. LGALS3 levels were highest in Intermediate and Unfavorable Cytogenetic groups. Pearson correlation of LGALS3 with the other proteins identified a distinct set of 23 proteins positively and 13 proteins negatively correlated with LGALS3 expression levels. LGALS3 positively correlated with the expression of anti-apoptotic BCL2 family member MCL-1. To validate RPPA results, OCI-AML3 cells were transduced with a lentivirus expressing shRNA against LGALS3 or with a control lentivirus expressing shRNA against a non-human gene (i.e. GFP). Suppression of LGALS3 in the cells results in reduced MCL-1 gene and protein expression. As reduction of LGALS3 by shRNA or inhibition of LGALS3 using GCS-100 suppresses phosphorylation of ERK, the mechanism how LGALS3 supports MCL-1 in the AML cells may involve ERK. OCI-AML3 LGALS3 shRNA cells were more sensitive to ABT-737 compared to control cells. RPPA also revealed that LGALS3 was strongly correlated with phosphorylated PKC delta and suppression of LGALS3 by shRNA reduced PKC delta phosphorylation levels implicating LGALS3 as a positive regulator of the kinase. While PKC delta is viewed foremost as a stress kinase, RPPA analysis of phosphorylated (i.e. active) PKC delta in AML patients reveals that elevated levels of active kinase is a prognostic factor associated with poor survival. The pro-survival role of PKC delta in AML may reflect its function as an AKT kinase or as a suppressor of ROS. RPPA also identified a novel positive association of LGALS3 with CD74. OCI-AML3 cells with LGALS3 shRNA did not exhibit altered CD74 expression suggesting that CD74 is upstream of the galectin or possibly that an as yet unidentified protein serves as a common regulator of both LGALS3 and CD74. Stromal cell derived CD74 has been implicated as important in the AML microenvironment as a regulator of IL-8 production (Abdul-Aziz et al Cancer Research 2016) but the role for CD74 in AML blast cells is not clear. CD74 modulates CD44 function so the protein could participate in signaling cascades involving the receptor. Pearson correlation with the other proteins analyzed identified 23 proteins including CD44 and LGALS3 that were positively correlated with CD74 and 8 proteins that were negatively associated with CD74. Though LGALS3 was not positively correlated with CD44, the galectin could potentially modulate CD44 function by influencing its surface expression as LGALS3 is involved in trafficking CD44 to the cell surface. The protein associations of LGALS3 and CD74 were used to assemble protein clusters that were then used for analysis of patient survival. LGALS3 and CD74 were collectively analyzed as part of a group of T-cell regulating proteins analyzed on this array. Within that group 4 different expression patterns (clusters) were defined by hierarchical clustering with LGALS3 being active in 2 of them. The protein cluster identifying active LGALS3 was prognostic for shorter remission duration. AML patients who exhibited both LGALS3 and CD74 active clusters exhibited very poor survival characteristics. For AML patients with both LGALS3 and CD74 active protein cluster, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. These data suggest that LGALS3 supports AML cell survival via diverse pathways including positive regulation of MCL-1 and PKC delta and support of a CD74 protein network that identifies a new high risk AML population. It is our hope that strategies targeting LGALS3 and CD74 can be developed to help this new at risk population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Microalgae Antiviral Activity and Their Bioactive Compounds.

During the last year, science has been focusing on the research of antivirally active compounds overall after the SARS-CoV-2 pandemic, which caused a great amount of deaths and the downfall of the economy in 2020. Photosynthetic organisms such as microalgae are known to be a reservoir of bioactive secondary metabolites; this feature, coupled with the possibility of achieving very high biomass levels without excessive energetic expenses, make microalgae worthy of attention in the search for new molecules with antiviral effects. In this work, the antiviral effects of microalgae against some common human or animal viruses were considered, focusing our attention on some possible effects against SARS-CoV-2. We summed up the data from the literature on microalgae antiviral compounds, from the most common ones, such as lectins, polysaccharides and photosynthetic pigments, to the less known ones, such as unidentified proteins. We have discussed the effects of a microalgae-based genetic engineering approach against some viral diseases. We have illustrated the potential antiviral benefits of a diet enriched in microalgae.

Osimertinib-resistant NSCLC cells activate ERBB2 and YAP/TAZ and are killed by neratinib

We performed additional mechanistic analyses to redefine neratinib biology and determined the mechanisms by which the multi-kinase inhibitor neratinib interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing either mutant KRAS (G12S; Q61H; G12A; G12C) or mutant NRAS (Q61K) or mutant ERBB1 (L858R; L858R T790M; exon 19 deletion). Neratinib rapidly reduced KRASG12V and RAC1G12V nanoclustering which was followed by KRASG12V, but not RAC1G12V, being extensively mislocalized away from the plasma membrane. This correlated with reduced levels of, and reorganized membrane localization of phosphatidylserine and cholesterol. Reduced nanoclustering was not associated with inactivation of ERBB1, Merlin or Ezrin. The drug combination killed cells expressing mutant KRAS, NRAS or mutant ERBB1 proteins. Afatinib or osimertinib resistant cells were killed with a similar efficacy to non-resistant cells. Compared to osimertinib-resistant cells, sensitive cells had less ERBB2 Y1248 phosphorylation. In osimertinib resistant H1975 cells, the drug combination was less capable of inactivating AKT, mTOR, STAT3, STAT5, ERK1/2 whereas it gained the ability to inactivate ERBB3. In resistant H1650 cells, the drug combination was less capable of inactivating JAK2 and STAT5. Sensitive cells exhibited elevated basal phosphorylation of YAP and TAZ. In resistant cells, portions of YAP and TAZ were localized in the nucleus. [Neratinib + pemetrexed] increased phosphorylation of YAP and TAZ, caused their nuclear exit, and enhanced ERBB2 degradation. Thus, neratinib targets an unidentified protein whose functional inhibition directly results in RAS inactivation and tumor cell killing. Our data prove that, albeit indirectly, oncogenic RAS proteins are druggable by neratinib.

Lack of ER Stress in NHERF1‐Deficient Proximal Tubule Cells Exhibiting Aberrant Npt2a Localization

Background NHERF1 (Na+-hydrogen exchanger regulatory factor isoform 1) anchors Npt2a (Na+-dependent phosphate cotransporter type IIa) in the brush border membrane (BBM) of proximal tubules through its cross-linking of Npt2a with the actin-filament binding protein, Ezrin. In the absence of NHERF1, Npt2a accumulates aberrantly in a subapical compartment. We have demonstrated that Npt2a associates with NHERF1 in the ER/Golgi as well as the BBM, suggesting a critical role for NHERF1 in the forward trafficking of Npt2a from the ER/Golgi to the BBM. Because increased levels of ER stress have been associated with intracellular accumulation of mis-localized proteins, we hypothesized that NHERF1 deficient proximal tubule cells may experience chronic ER stress associated with mis-localization of Npt2a. Methods We measured protein expression of four ER stress markers (phosphorylated eIF2α, GRP78, GRP94, and p58IPK) in kidney cortex isolated from C57BL/6J wild type (WT) mice and their NHERF1 knock-out (KO) littermates by immunoblot and mRNA levels by RNAseq. We expressed a fluorescently labeled WT Npt2a and NHERF1 or a Npt2a/NHERF1 chimera substituting the C terminal PDZ binding motif of Npt2a with the ERM (Ezrin-Radixin-Moesin) binding domain of NHERF1 in human embryonic kidney (HEK), opossum kidney (OK), and NHERF1-deficient opossum kidney (OKH) cells. We compared WT vs chimera protein expression by confocal microscopy and protein function by radiolabeled phosphate uptake. Results We found no differences in kidney cortex protein or mRNA expression of these ER stress markers between WT and KO animals. Confocal microscopy of the Npt2a chimera expressed in HEK, OK, and OKH cells exhibited enhanced apical membrane localization when compared to WT Npt2a in all three cell types. However, no increase in radiolabeled phosphate uptake was observed between cells expressing the Npt2a chimera or wild type Npt2a. Conclusions The lack of an ER stress response suggests that despite association between NHERF1 and Npt2a in the ER/Golgi, NHERF1 is not required for normal Npt2a folding. Enhanced apical expression of the Npt2a chimera confirms the essential role of Ezrin in BBM localization of Npt2a, however, appropriate Npt2a function requires the presence of more than the ERM binding domain of NHERF1, possibly involving the association of other unidentified proteins.

Detecting Protein Communities in Native Cell Extracts by Machine Learning: A Structural Biologist's Perspective.

Native cell extracts hold great promise for understanding the molecular structure of ordered biological systems at high resolution. This is because higher-order biomolecular interactions, dubbed as protein communities, may be retained in their (near-)native state, in contrast to extensively purifying or artificially overexpressing the proteins of interest. The distinct machine-learning approaches are applied to discover protein–protein interactions within cell extracts, reconstruct dedicated biological networks, and report on protein community members from various organisms. Their validation is also important, e.g., by the cross-linking mass spectrometry or cell biology methods. In addition, the cell extracts are amenable to structural analysis by cryo-electron microscopy (cryo-EM), but due to their inherent complexity, sorting structural signatures of protein communities derived by cryo-EM comprises a formidable task. The application of image-processing workflows inspired by machine-learning techniques would provide improvements in distinguishing structural signatures, correlating proteomic and network data to structural signatures and subsequently reconstructed cryo-EM maps, and, ultimately, characterizing unidentified protein communities at high resolution. In this review article, we summarize recent literature in detecting protein communities from native cell extracts and identify the remaining challenges and opportunities. We argue that the progress in, and the integration of, machine learning, cryo-EM, and complementary structural proteomics approaches would provide the basis for a multi-scale molecular description of protein communities within native cell extracts.

TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I

Human mitochondrial ATP synthase is a molecular machine with a rotary action bound in the inner organellar membranes. Turning of the rotor, driven by a proton motive force, provides energy to make ATP from ADP and phosphate. Among the 29 component proteins of 18 kinds, ATP6 and ATP8 are mitochondrial gene products, and the rest are nuclear gene products that are imported into the organelle. The ATP synthase is assembled from them via intermediate modules representing the main structural elements of the enzyme. One such module is the c8-ring, which provides the membrane sector of the enzyme's rotor, and its assembly is influenced by another transmembrane (TMEM) protein, TMEM70. We have shown that subunit c interacts with TMEM70 and another hitherto unidentified mitochondrial transmembrane protein, TMEM242. Deletion of TMEM242, similar to deletion of TMEM70, affects but does not completely eliminate the assembly of ATP synthase, and to a lesser degree the assembly of respiratory enzyme complexes I, III, and IV. Deletion of TMEM70 and TMEM242 together prevents assembly of ATP synthase and the impact on complex I is enhanced. Removal of TMEM242, but not of TMEM70, also affects the introduction of subunits ATP6, ATP8, j, and k into the enzyme. TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I. The interactions of TMEM70 and TMEM242 with MCIA could be part of either the assembly of ATP synthase and complex I or the regulation of their levels.

Proteomic characterization of the pellicle of Toxoplasma gondii

Toxoplasma gondii is one of the most successful intracellular parasites in the world. The dynamic, adhesion, invasion, and even replication capabilities of Toxoplasma are based on dynamic machinery located in the pellicle, a three membrane complex that surrounds the parasite. Among the proteins that carry out these processes are inner membrane complex (IMC) proteins, gliding-associated proteins (GAP), diverse myosins, actin, tubulin, and SRS proteins. Despite the importance of the pellicle, the knowledge of its composition is limited. Broad protein identification from an enriched pellicle fraction was obtained by independent digestion with trypsin and chymotrypsin and quantified by mass spectrometry. By trypsin digestion, 548 proteins were identified, while by chymotrypsin digestion, additional 22 proteins were identified. Besides, a group of “sequences related to SAG1” proteins (SRS) were detected together with unidentified new proteins. From identified SRS proteins, SRS51 was chosen for analysis and modeling as its similarities with crystallized adhesion proteins, exhibiting the presence of a spatial groove that is apparently involved in adhesion and cell invasion. As SRS proteins have been reported to be involved in the activation of the host's immune response, further studies could consider them as targets in the design of vaccines or of drugs against Toxoplasma. SignificanceTo date, the proteomic composition of the pellicle of Toxoplasma is unknown. Most proteins reported in Toxoplasma pellicle have been poorly studied, and many others remain unidentified. Herein, a group of new SRS proteins is described. Some SRS proteins previously described from pellicle fraction have adhesion properties to the host cell membrane, so their study would provide data related to invasion mechanism and to open possibilities for considering them as targets in the design of immunoprotective strategies or the design of new pharmacological treatments.

Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression

Glutathione (GSH) is an important antioxidant that plays a critical role in neuroprotection. GSH depletion in neurons induces oxidative stress and thereby promotes neuronal damage, which in turn is regarded as a hallmark of the early stage of neurodegenerative diseases. The neuronal GSH level is mainly regulated by cysteine transporter EAAC1 and its inhibitor, GTRAP3-18. In this study, we found that the GTRAP3-18 level was increased by up-regulation of the microRNA miR-96-5p, which was found to decrease EAAC1 levels in our previous study. Since the 3’-UTR region of GTRAP3-18 lacks the consensus sequence for miR-96-5p, an unidentified protein should be responsible for the intermediate regulation of GTRAP3-18 expression by miR-96-5p. Here, we discovered that RNA-binding protein NOVA1 functions as an intermediate protein for GTRAP3-18 expression via miR-96-5p. Moreover, we show that intra-arterial injection of a miR-96-5p-inhibiting nucleic acid to living mice by a drug delivery system using microbubbles and ultrasound decreased the level of GTRAP3-18 via NOVA1 and increased the levels of EAAC1 and GSH in the dentate gyrus of the hippocampus. These findings suggest that the delivery of a miR-96-5p inhibitor to the brain would efficiently increase the neuroprotective activity by increasing GSH levels via EAAC1, GTRAP3-18 and NOVA1.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s).

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 &amp;gt; 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).