Protein Transport Research Articles

Transcriptome analysis of the degradation process of organic nitrogen by two heterotrophic nitrifying and aerobic denitrifying bacteria.

The heterotrophic nitrification aerobic denitrification bacteria (HNDS) can perform nitrification and denitrification at the same time. Two HNDS strains, Achromobacter sp. HNDS-1 and Enterobacter sp. HNDS-6 which exhibited an amazing ability to solution nitrogen (N) removal have been successfully isolated from paddy soil in our lab. When peptone or ammonium sulfate as sole N source, no significant difference in gene expression related to nitrification and denitrification of the strains was found according to the transcriptome analysis. The expression of phosphomethylpyrimidine synthase (thiC), ABC transporter substrate-binding protein, branched-chain amino acid ABC transporter substrate-binding protein, and RNA polymerase (rpoE) in HNDS-1 were significantly upregulated when used peptone as N source, while the expression of exopolysaccharide production protein (yjbE), RNA polymerase (rpoC), glutamate synthase (gltD) and ABC-type branched-chain amino acid transport systems in HNDS-6 were significantly upregulated. This indicated that these two strains are capable of using organic N and converting it into NH4+-N, then utilizing NH4+-N to synthesize amino acids and proteins for their own growth, and strain HNDS-6 can also remove NH4+-N through nitrification and denitrification.

An extended interaction site determines binding between AP180 and AP2 in clathrin mediated endocytosis

The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution. We find that the 70 residue-long site determines the overall interaction between AP180 and AP2 in a dynamic equilibrium between its bound and unbound states, while weaker binding sites contribute to the overall affinity at much higher concentrations of AP2. Our data suggest that this particular interaction site might play a central role in recruitment of adaptors to the clathrin coated pit, whereas more transient and promiscuous interactions allow reshaping of the interaction network until cargo uptake inside a coated vesicle.

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans

Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1−/− males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood–testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility.

Structural Basis for the Interaction between the Ezrin FERM-Domain and Human Aquaporins.

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.

In Vivo Proximity Cross-Linking and Immunoprecipitation of Cell Wall Epitopes Identify Proteins Associated with the Biosynthesis of Matrix Polysaccharides.

Identification of proteins involved in cell wall matrix polysaccharide biosynthesis is crucial to understand plant cell wall biology. We utilized in vivo cross-linking and immunoprecipitation with cell wall antibodies that recognized xyloglucan, xylan, mannan, and homogalacturonan to capture proteins associated with matrix polysaccharides in Arabidopsis protoplasts. The use of cross-linkers allowed us to capture proteins actively associated with cell wall polymers, including those directly interacting with glycans via glycan-protein (GP) cross-linkers and those associated with proteins linked to glycans via a protein-protein (PP) cross-linker. Immunoprecipitations led to the identification of 65 Arabidopsis protein IDs localized in the Golgi, ER, plasma membrane, and others without subcellular localization data. Among these, we found several glycosyltransferases directly involved in polysaccharide synthesis, along with proteins related to cell wall modification and vesicle trafficking. Protein interaction networks from DeepAraPPI and AtMAD databases showed interactions between various IDs, including those related to cell-wall-associated proteins and membrane/vesicle trafficking proteins. Gene expression and coexpression analyses supported the presence and relevance of the proteins to the cell wall processes. Reverse genetic studies using T-DNA insertion mutants of selected proteins revealed changes in cell wall composition and saccharification, further supporting their potential roles in cell wall biosynthesis. Overall, our approach represents a novel approach for studying cell wall polysaccharide biosynthesis and associated proteins, providing advantages over traditional immunoprecipitation techniques. This study provides a list of putative proteins associated with different matrix polysaccharides for further investigation and highlights the complexity of cell wall biosynthesis and trafficking within plant cells.

TFG regulates inner COPII coat recruitment to facilitate anterograde secretory protein transport.

Coat protein complex II (COPII) governs the initial steps of biosynthetic secretory protein transport from the endoplasmic reticulum (ER), facilitating the movement of a wide variety of cargoes. Here, we demonstrate that Trk-fused gene (TFG) regulates the rate at which inner COPII coat proteins are concentrated at ER subdomains. Specifically, in cells lacking TFG, the GTPase-activating protein (GAP) Sec23 accumulates more rapidly at budding sites on the ER as compared with control cells, potentially altering the normal timing of GTP hydrolysis on Sar1. Under these conditions, anterograde trafficking of several secretory cargoes is delayed, irrespective of their predicted size. We propose that TFG controls the local, freely available pool of Sec23 during COPII coat formation and limits its capacity to prematurely destabilize COPII complexes on the ER. This function of TFG enables it to act akin to a rheostat, promoting the ordered recruitment of Sec23, which is critical for efficient secretory cargo export.

The deubiquitinating enzymes-related signature predicts the prognosis and immunotherapy response in breast cancer.

Breast cancer is a prevalent disease that has a dismal prognosis for patients and a bad outlook for treatments. Ubiquitination is a reversible biological process that regulates protein production and degradation, as well as plays a vital role in protein transport, localization, and biological activity. We obtained the breast cancer patient sample data and used a machine learning technique to create a novel index called Deubiquitinating enzyme related index (DUBRI) by gathering genes associated to deubiquitinating enzymes. Based on DUBRI, we systematically analyze patients' prognosis, clinical characteristics, tumor immune microenvironment, chemotherapy response and immunotherapy response. Finally, the function of OTUB2 was explored in breast cancer cells. DUBRI, which consists of five deubiquitinating enzyme genes (OTUB2, USP41, MINDY2, YOD1, and PSMD7), is a reliable predictor of survival in breast cancer patients. We found that the high DUBRI group presented higher levels of immune cell infiltration. We performed molecular docking prediction of core target proteins in deubiquitinating enzymes. In vitro experiments verified that knockdown of OTUB2 could inhibit the proliferation and migration of breast cancer. The DUBRI discovered in this research may effectively evaluate the outlook of breast cancer patients and identify groups of patients who would gain advantages from immunotherapy, offering vital knowledge for the future targeted treatment of breast cancer patients.

MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.

N-Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg2+ influx in conditions of high glucose or impaired respiratory chain function.

N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher Mr MRS2 bands gel-shifted to lower Mr and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher Mr MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+ under conditions of glucose excess or mitochondrial bioenergetic impairment.

AZGs: a new family of cytokinin transporters.

Cytokinins (CKs) are phytohormones structurally similar to purines that play important roles in various aspects of plant physiology and development. The local and long-distance distribution of CKs is very important to control their action throughout the plant body. Over the past decade, several novel CK transporters have been described, many of which have been linked to a physiological function rather than simply their ability to transport the hormone in vitro. Purine permeases, equilibrative nucleotide transporters and ATP-binding cassette transporters are involved in the local and long-range distribution of CK. In addition, members of the Arabidopsis AZA-GUANINE RESISTANT (AZG) protein family, AZG1 and AZG2, have recently been shown to mediate CK uptake at the plasma membrane and endoplasmic reticulum. Despite sharing ∼50% homology, AZG1 and AZG2 have unique transport mechanisms, tissue-specific expression patterns, and subcellular localizations that underlie their distinct physiological functions. AZG2 is expressed in a small group of cells in the overlying tissue around the lateral root primordia, where its expression is induced by auxins and it is involved in the regulation of lateral root growth. AZG1 is ubiquitously expressed, with high levels in the division zone of the root apical meristem. Here, it binds and stabilises the auxin efflux carrier PIN1, thereby shaping root architecture, particularly under salt stress. This review highlights the latest findings on the protein properties, transport mechanisms and cellular functions of this new family of CK transporters and discusses perspectives for future research in this field.

Trafficking of adhesion and aggregation-modulating proteins during the early stages of Dictyostelium development

The social amoeba Dictyostelium discoideum has been studied for close to a century to better understand conserved cellular and developmental processes. The life cycle of this model eukaryote is composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. When starved, individual cells undergo chemotactic aggregation to form multicellular mounds that develop into slugs. Terminal differentiation of cells within slugs forms fruiting bodies, each composed of a stalk that supports a mass of viable spores that germinate and restart the life cycle when nutrients become available. Calcium-dependent cell adhesion protein A (CadA) and countin (CtnA) are two proteins that regulate adhesion and aggregation, respectively, during the early stages of D. discoideum development. While the functions of these proteins have been well-studied, the mechanisms regulating their trafficking are not fully understood. In this study, we reveal pathways and cellular components that regulate the intracellular and extracellular amounts of CadA and CtnA during aggregation. During growth and starvation, CtnA localizes to cytoplasmic vesicles and punctae. We show that CtnA is glycosylated and this post-translational modification is required for its secretion. Upon autophagy induction, a signal peptide for secretion facilitates the release of CtnA from cells via a pathway involving the μ subunit of the AP3 complex (Apm3) and the WASP and SCAR homolog, WshA. Additionally, CtnA secretion is negatively regulated by the D. discoideum orthologs of the human non-selective cation channel mucolipin-1 (Mcln) and sorting receptor sortilin (Sort1). As for CadA, it localizes to the cell periphery in growth-phase and starved cells. The intracellular and extracellular amounts of CadA are modulated by autophagy genes (atg1, atg9), Apm3, WshA, and Mcln. We integrate these data with previously published findings to generate a comprehensive model summarizing the trafficking of CadA and CtnA in D. discoideum. Overall, this study enhances our understanding of protein trafficking during D. discoideum aggregation, and more broadly, provides insight into the multiple pathways that regulate protein trafficking and secretion in all eukaryotes.

Short transmembrane domains target type II proteins to the Golgi apparatus and type I proteins to the endoplasmic reticulum.

Transmembrane domains (TMDs) contain information targeting membrane proteins to various compartments of the secretory pathway. In previous studies, short or hydrophilic TMDs have been shown to target membrane proteins either to the endoplasmic reticulum (ER) or to the Golgi apparatus. However, the basis for differential sorting to the ER and to the Golgi apparatus remained unclear. To clarify this point, we quantitatively analyzed the intracellular targeting of a collection of proteins exhibiting a single TMD. Our results reveal that membrane topology is a major targeting element in the early secretory pathway: type I proteins with a short TMD are targeted to the ER, and type II proteins to the Golgi apparatus. A combination of three features accounts for the sorting of simple membrane proteins in the secretory pathway: membrane topology, length and hydrophilicity of the TMD, and size of the cytosolic domain. By clarifying the rules governing sorting to the ER and to the Golgi apparatus, our study could revive the search for sorting mechanisms in the early secretory pathway.

A deep learning-driven discovery of berberine derivatives as novel antibacterial against multidrug-resistant Helicobacter pylori

Helicobacter pylori (H. pylori) is currently recognized as the primary carcinogenic pathogen associated with gastric tumorigenesis, and its high prevalence and resistance make it difficult to tackle. A graph neural network-based deep learning model, employing different training sets of 13,638 molecules for pre-training and fine-tuning, was aided in predicting and exploring novel molecules against H. pylori. A positively predicted novel berberine derivative 8 with 3,13-disubstituted alkene exhibited a potency against all tested drug-susceptible and resistant H. pylori strains with minimum inhibitory concentrations (MICs) of 0.25–0.5 μg/mL. Pharmacokinetic studies demonstrated an ideal gastric retention of 8, with the stomach concentration significantly higher than its MIC at 24 h post dose. Oral administration of 8 and omeprazole (OPZ) showed a comparable gastric bacterial reduction (2.2-log reduction) to the triple-therapy, namely OPZ + amoxicillin (AMX) + clarithromycin (CLA) without obvious disturbance on the intestinal flora. A combination of OPZ, AMX, CLA, and 8 could further decrease the bacteria load (2.8-log reduction). More importantly, the mono-therapy of 8 exhibited comparable eradication to both triple-therapy (OPZ + AMX + CLA) and quadruple-therapy (OPZ + AMX + CLA + bismuth citrate) groups. SecA and BamD, playing a major role in outer membrane protein (OMP) transport and assembling, were identified and verified as the direct targets of 8 by employing the chemoproteomics technique. In summary, by targeting the relatively conserved OMPs transport and assembling system, 8 has the potential to be developed as a novel anti-H. pylori candidate, especially for the eradication of drug-resistant strains.

Analysis of the radiation effect on the activity of thyroid blood transport systems: connection with the features of thyroid status

Thyroid-binding proteins play an important role in regulating thyroid status. However, the question of the radiation effect on the functional state of thyroid blood transport systems remains insufficiently studied. The aim of the work was to assess the indicators of thyroid status and thyroid blood transport in persons exposed to radiation as a result of the Chernobyl accident. The task of the work was also to analyze the results of an oral test for thyroxine pharmacokinetics in patients with thyroid carcinoma who received radioiodine therapy. Screening studies conducted 5−6 years after the Chernobyl accident indicate the radiation effect on the free thyroxine content in the blood and the functional activity of thyroid protein transport in children and adolescents living in the territories of the Khoiniki district of the Gomel region. The dose-dependent effect of an absorbed dose of 131I was also detected in children and adolescents displaced from the 30 km zone of the Chernobyl accident. The results of an oral test for thyroxine pharmacokinetics demonstrate an increase in the free T4 content against the background of a stable total T4 fraction in patients with thyroid carcinoma who received high dose loads as a result of radioiodine therapy. The conducted studies confirm the possibility of the influence of a high dose of 131I on thyroid blood transport systems and on the ratio of free and bound fractions of thyroid hormones in the blood.

Sorting of GPI-anchored proteins at the trypanosome surface is independent of GPI insertion signals

The segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) to distinct domains on the plasma membrane of eukaryotic cells is important for their correct cellular function, but the mechanisms by which GPI-APs are sorted are yet to be fully resolved. An extreme example of this is in African trypanosomes, where the major surface glycoprotein floods the whole cell surface while most GPI-APs are retained in a specialised domain at the base of the flagellum. One possibility is that anchor attachment signals direct differential sorting of proteins. To investigate this, we fused a monomeric reporter to the GPI-anchor insertion signals of trypanosome proteins that are differentially sorted on the plasma membrane. Fusions were correctly anchored by GPI, post-translationally modified, and routed to the plasma membrane, but this delivery was independent of retained signals upstream of the ω site. Instead, ω−minus signal strength appears key to efficacy of GPI addition and to GPI-AP cellular level. Thus, at least in this system, sorting is not encoded at the time of GPI anchor addition or in the insertion sequence retained in processed proteins. We discuss these findings in the context of previously proposed models for sorting mechanisms in trypanosomes.

The journey of STING: Guiding immune signaling through membrane trafficking

Stimulator of Interferon Genes (STING) serves as a pivotal mediator in the innate immune signaling pathway, transducing signals from various DNA receptors and playing a crucial role in natural immune processes. During cellular quiescence, STING protein resides in the endoplasmic reticulum (ER), and its activation typically occurs through the cGAS-STING signaling pathway. Upon activation, STING protein is transported to the Golgi apparatus, thereby initiating downstream signaling cascades. Vesicular transport serves as the primary mechanism for STING protein trafficking between the ER and Golgi apparatus, with COPII mediating anterograde transport from the ER to Golgi apparatus, while COPI is responsible for retrograde transport. Numerous factors influence these transport processes, thereby exerting either promoting or inhibitory effects on STING protein expression. Upon reaching the Golgi apparatus, to prevent over-activation, STING protein is transported to post-Golgi compartments for degradation. In addition to the conventional lysosomal degradation pathway, ESCRT has also been identified as one of the degradation pathways for STING protein. This review summarizes the recent findings on the membrane trafficking pathways of STING, highlighting their contributions to the regulation of cytokine production, the activation of immune cells, and the coordination of immune signaling pathways.

Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.

A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here, we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the endoplasmic reticulum (ER) exit sites and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12, and Sec24, and participates in coat protein complex II vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

Multiple types of nuclear localization signals in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite that belongs to the Amoebozoa supergroup whose study related to the nucleocytoplasmic transport of proteins through the nucleus is poorly studied. In this work, we have performed in silico predictions of the potential nuclear localization signals (NLS) corresponding to the proteome of 8201 proteins from Entamoeba histolytica annotated in the AmoebaDB database. We have found the presence of monopartite nuclear localization signals (MNLSs), bipartite nuclear localization signals (BNLSs), and non-canonical monopartite NLSs with lengths exceeding 20 amino acid residues. Additionally, we detected a new type of NLS consisting of multiple juxtaposed bipartite NLSs (JNLSs) that have not been described in any eukaryotic organism. Also, we have generated consensus sequences for the nuclear import of proteins with the NLSs obtained. Docking experiments between EhImportin α and an MNLS, BNLS, and JNLS outlined the interacting residues between the Importin and cargo proteins, emphasizing their putative roles in nuclear import. By transfecting HA-tagged protein constructs, we assessed the nuclear localization of MNLS (U1A and U2AF1), JMNLS (U2AF2), and non-canonical NLS (N-terminus of Pol ll) in vivo. Our data provide the basis for understanding the nuclear transport process in E. histolytica.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson's disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.