Translocation Domain Research Articles

Development of inhibition ELISA to detect antibody-induced failure of botulinum toxin a therapy in cosmetic indications

Secondary treatment failure (STF) of botulinum toxin A (BoNT/A) therapy in cosmetic indication has been postulated as production of antibody against active sites of BoNT/A in unresponsive patients. To prove of concept, detection of anti-BoNT/A antibody is required, however, current enzyme-linked immunosorbent assay (ELISA) detects human IgGs against whole BoNT/A molecule. We developed an inhibition ELISA to quantify antibodies bound to the active sites of BoNT/A using three mouse monoclonal antibodies targeting translocation domain, receptor binding site and catalytic domain of BoNT/A prior to processing ELISA to detect human IgG (hIgG) against BoNT/A. Adults naïve to BoNT/A, or treated and responsive (toxin-response), or treated but unresponsive (toxin-tolerance) were recruited. Detection of hIgG revealed that naïve volunteers had basal level of hIgG against whole BoNT/A, whereas its level was significantly lower than those hIgG in BoNT/A-exposed cohorts. Higher anti-BoNT/A levels in sera from volunteers ever-exposed to BoNT/A indicates that BoNT/A may provoke immune responses in BoNT/A-treated cohorts. Inhibition ELISA demonstrated that levels of BoNT/A-specific hIgG in tolerance patients had a dramatic decrease in mouse monoclonal antibody blockage, suggesting presence of hIgG specific to BoNT/A's three active sites in STF patients. Therefore, our ELISA detected hIgG against whole BoNT/A protein and BoNT/A active sites suggesting that human antibodies may cause STF. To compare with frontalis test, our inhibition ELISA provided good accuracy at 83.1% (50% sensitivity and 89.9% specificity). Our test may help clinicians to diagnose possibility of STF and also to monitor immune status against BoNT/A.

Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model

Engagement of programmed death 1 receptor (PD-1) and its ligand PD-L1/2 induces a signal transduction pathway that inhibits the activity of tumor-infiltrating cytotoxic T lymphocytes and promotes tumor growth and metastasis. Antibodies blocking PD-1 or PD-L1 can restore antitumor T cell responses and cause long-term remission in a subset of cancer patients with advanced or refractory tumors. In this study, we asked whether PD-L1 vaccination could confer tumor control in mouse tumor models. To address the central tolerance toward self-molecules, we fused the extracellular domain of PD-L1 (PD-L1E) to the C-terminal of the translocation domain of diphtheria toxin (DTT). DTT is able to elicit CD4+ T cell responses required for inducing robust immune responses against self-molecules. The fusion molecule is called DPDL1E. When formulated with incomplete Freund’s adjuvant (IFA), DPDL1E elicited robust immune responses biased toward the Th1 type and inhibited tumor growth in both preventive and therapeutic mouse tumor models. We further showed that the anti-DPDL1E sera blocked PD-L1 binding to PD-1 in vitro. The DPDL1E vaccination increased the levels of tumor-infiltrating T lymphocytes (TILs) and reduced the levels of myeloid-derived suppressor cells (MDSCs) as well as exhausted LAG3+PD-1+ CD8+ T cells. All of these data suggest that DPDL1E vaccination reverses the suppressive phenotype of the tumor microenvironment and that it is a promising strategy for cancer therapy.

Type V Secretion Systems: An Overview of Passenger Domain Functions.

Bacteria secrete proteins for different purposes such as communication, virulence functions, adhesion to surfaces, nutrient acquisition, or growth inhibition of competing bacteria. For secretion of proteins, Gram-negative bacteria have evolved different secretion systems, classified as secretion systems I through IX to date. While some of these systems consist of multiple proteins building a complex spanning the cell envelope, the type V secretion system, the subject of this review, is rather minimal. Proteins of the Type V secretion system are often called autotransporters (ATs). In the simplest case, a type V secretion system consists of only one polypeptide chain with a β-barrel translocator domain in the membrane, and an extracellular passenger or effector region. Depending on the exact domain architecture of the protein, type V secretion systems can be further separated into sub-groups termed type Va through e, and possibly another recently identified subtype termed Vf. While this classification works well when it comes to the architecture of the proteins, this is not the case for the function(s) of the secreted passenger. In this review, we will give an overview of the functions of the passengers of the different AT classes, shedding more light on the variety of functions carried out by type V secretion systems.

Orphan designation: Recombinant protein consisting of modified human growth hormone releasing hormone and the translocation and endopeptidase domains of botulinum toxin serotype D, Treatment of acromegaly

Orphan designation: Recombinant protein consisting of modified human growth hormone releasing hormone and the translocation and endopeptidase domains of botulinum toxin serotype D, Treatment of acromegaly

A Hierarchical Approach to Predict Conformation-Dependent Histidine Protonation States in Stable and Flexible Proteins.

Solution acidity measured by pH is an important environmental factor that affects protein structure. It influences the protonation state of protein residues, which in turn may be coupled to protein conformational changes, unfolding, and ligand binding. It remains difficult to compute and measure the p Ka of individual residues, as well as to relate them to pH-dependent protein transitions. This paper presents a hierarchical approach to compute the p Ka of individual protonatable residues, specifically histidines, coupled with underlying structural changes of a protein. A fast and efficient free energy perturbation (FEP) algorithm has also been developed utilizing a fast implementation of standard molecular dynamics (MD) algorithms. Specifically, a CUDA version of the AMBER MD engine is used in this paper. Eight histidine p Ka's are computed in a diverse set of pH stable proteins to demonstrate the proposed approach's utility and assess the predictive quality of the AMBER FF99SB force field. A reference molecule is carefully selected and tested for convergence. A hierarchical approach is used to model p Ka's of the six histidine residues of the diphtheria toxin translocation domain (DTT), which exhibits a diverse ensemble of individual conformations and pH-dependent unfolding. The hierarchical approach consists of first sampling equilibrium conformational ensembles of a protein with protonated and neutral histidine residues via long equilibrium MD simulations (Flores-Canales, J. C.; et al. bioRxiv, 2019, 572040). A clustering method is then used to identify sampled protein conformations, and p Ka's of histidines in each protein conformation are computed. Finally, an ensemble averaging formalism is developed to compute weighted average histidine p Ka's. These can be compared with an apparent experimentally measured p Ka of the DTT protein and thus allows us to propose a mechanism of pH-dependent unfolding of the DTT protein.

In vitro assembly of Haemophilus influenzae adhesin transmembrane domain and studies on the electrostatic repulsion at the interface.

Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family, and it mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia is composed of the passenger domain, which is a virulence factor, and the translocator domain, which anchors the passenger domain into the outer membrane. The Hia transmembrane domain forms a transmembrane β-barrel of 12 β-strands, four of which are provided from each subunit. The β-barrel has a pore that is traversed by three α-helices, one of which is provided from each subunit. This domain has a unique arginine arrangement inside the β-barrel. The side chains of the arginine residues protrude from the β-strands of three subunits toward the center of the barrel and are close to each other. Mutation of this arginine residue revealed the importance of the electrostatic repulsion between the three arginines. Electrostatic repulsion is considered to prevent misfolding and/or misassembly. The arginine clusters at the interface were found in several proteins and might generally play an important role in the assembly of the oligomer.

Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440.

Summary Pseudomonas putida can be used as a host for the autotransporter‐mediated surface display of enzymes (autodisplay), resulting in whole‐cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter‐mediated secretion depends on the N‐terminal signal peptide as well as on the C‐terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole‐cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12‐fold enhanced to 638 mU ml−1 OD −1 compared with the signal peptide of V. cholerae CtxB (52 mU ml−1 OD −1). This positive effect was confirmed with a β‐glucosidase as a second example enzyme. Here, cells expressing the protein with N‐terminal OprF signal peptide showed more than fourfold higher β‐glucosidase activity (181 mU ml−1 OD −1) than with the CtxB signal peptide (42 mU ml−1 OD −1). SDS‐PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.

Targeted Protein Degradation through Cytosolic Delivery of Monobody Binders Using Bacterial Toxins.

Monobodies are small engineered binding proteins that, upon expression in cells, can inhibit signaling of cytosolic oncoproteins with outstanding selectivity. Efficacy may be further increased by inducing degradation of monobody targets through fusion to the von Hippel–Lindau (VHL) substrate receptor of the Cullin2-E3 ubiquitin ligase complex. However, potential therapeutic use is currently limited, because of the inability of monobody proteins to cross cellular membranes. Here, we use a chimeric bacterial toxin, composed of the Shiga-like toxin B (Stx2B) subunit and the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA-II) for delivery of VHL–monobody protein fusions to target endogenous tyrosine kinases in cancer cells. Depending on the expression of the Stx2B receptor Gb3 on the cell surface, we show that monobodies are taken up by an endocytic route, but are not degraded in lysosomes. Delivery of monobodies fused to a nuclear localization signal resulted in accumulation in the nucleus, thereby indirectly, but unequivocally, demonstrating cytosolic delivery. Delivery of VHL fused to monobodies targeting the Lck tyrosine kinase in T-cells resulted in reduced Lck protein levels, which was dependent on the expression of Gb3. This led to the inhibition of proximal signaling events downstream of the T-cell receptor complex. This work provides a prime example of the delivery of a stoichiometric protein inhibitor of an endogenous target protein to cells and inducing its degradation without the need of genetic manipulation of target cells. It lays the foundation for further in vivo exploitation of this delivery system.

Investigating the uptake mechanism of S type pyocins

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen with a high mortality and morbidity rate. It has many mechanisms of resistance to antibiotics, which makes it hard to treat. Pyocin is a protein that is produced by P.aeruginosa that kills related strain bacteria. pyocin type S bind to ferrisiderophore receptors that uses the TonB system to translocate into the bacterial cell. The aim of this study was to express and purify receptor and translocation (R+T) domains of pyocin S1, S2 and S3 plus the TonB1 receptor and ToLAIII receptor of P.aeruginosa to determine if pyocins S uses these to transverse the inner membrane of target cells. IPTG was used to induce the protein in the expression and analyzed by SDS-PAGE gel, giving fragment size of 11 kDa (ToLAIII), 23 kDa (TonB1), 45 kDa (S2 R+T) and 79 kDa (S3 R+T). Protein purification was cried on the four proteins using affinity chromatography technique by His-tag in C-terminal of S2 and S3 R+T domain, N-terminal of TonB1 and ToLAIII proteins. In addition, Gel Filtration chromatography was used to further purify the proteins so that the interaction between them could be tested. This study confirmed that the proteins used is expressed and purified, so in the future study the gel filtration would be carried out for R+T domains of the other S pyocins and TonB1 protein so that the interaction would be tested between the proteins in the study.

A Self-Actuated Cellular Protein Delivery Machine.

Engineered bacterial cells have great promise to solve global problems, yet they are hampered by a lack of convenient strategy for controlled protein release. A well-controlled protein translocation through cellular membranes is essential for cell-based protein delivery. Here we have developed a controlled protein release system by programming a bacterial autotransporter system named Ag43. Ag43 protein is engineered by adding a protease digestion site between its translocation and cargo domains. Once it is displayed on the cell surface, we managed to release the cargo proteins in defined conditions by processing environmental signals. The protein release in terms of time and quantity can be controlled through changing the inducer conditions. We thought that the release system can be adopted for complex genetic circuitries due to its simplicity. We implemented the protein release system to develop a cellular device that is able to release proteins in a sequence response to ordered chemical signals. We envision that development of genetically controlled protein release systems will improve the applications of synthetic organisms in cell based therapies, especially for cases with a need for controlled protein release using the cues from the biological environment.

Notch signaling pathway mediates alveolar bone resorption in apical periodontitis

Apical periodontitis represents a chronic inflammatory process within periapical tissues, mostly caused by etiological agents of endodontic origin. Progressive bone resorption in the periapical region represents the hallmark of apical periodontitis and occurs as the consequence of interplay between polymicrobial infections and host response. The Notch signaling pathway is an evolutionary conserved cell-signaling system that plays an important role in a variety of cell functions including proliferation, differentiation and apoptosis. In recent years its involvement in bone homeostasis has attracted a significant consideration. We hypothesized that Notch signaling pathway, which has a complex interplay with proinflammatory cytokines and bone resorption regulators, contributes to alveolar bone resorption via increased Notch receptors on immune cell surface and stimulates Notch receptor intracellular domain (NICD) translocation into the nucleus. The potential benefit of medications aimed to down-regulate these pathways in apical periodontitis treatment remains to be assessed.

Role of Diffusion in Unfolding and Translocation of Multidomain Titin I27 Substrates by a Clp ATPase Nanomachine.

Degradation of multidomain substrate proteins (SPs) by AAA+ nanomachines takes place processively from the tagged terminal. Ring-shaped ATPase components, such as ClpY, apply repetitive mechanical forces to effect domain unfolding and translocation of polypeptide segments through a narrow central channel. We study these mechanisms through atomistic Langevin dynamics simulations of C-terminal-tagged SPs in allosteric ClpY cycles. We find that monomeric SPs are processed through single unfolding pathways and fast timescales, whereas multimeric SPs involve branched pathways and slower timescales. These distinct mechanisms are attributed to the slower rotational diffusion of the C-terminal domain in multidomain SPs that hinders access to the soft mechanical direction. In the geometry specific to laser optical tweezers experiments, involving a restrained SP N-terminal, a single unfolding pathway is found for both monomeric and tetrameric SPs as pulling is applied along the N-C direction. Non-native interactions modulate unfolding of unrestrained monomers by weakening the C-terminal interface but do not contribute significantly to unfolding of restrained SPs. On the basis of these results, we propose that the interplay of restricted SP dynamics and ATPase kinetics underlies partitioning of multidomain SPs into completely degraded products and undegraded fragments comprising folded domains.

Combination of Cell-Penetrating Peptides with Nanoparticles for Therapeutic Application: A Review.

Cell-penetrating peptides (CPPs), also known as protein translocation domains, membrane translocating sequences or Trojan peptides, are small molecules of 6 to 30 amino acid residues capable of penetrating biological barriers and cellular membranes. Furthermore, CPP have become an alternative strategy to overcome some of the current drug limitations and combat resistant strains since CPPs are capable of delivering different therapeutic molecules against a wide range of diseases. In this review, we address the recent conjugation of CPPs with nanoparticles, which constitutes a new class of delivery vectors with high pharmaceutical potential in a variety of diseases.

Evaluating Potential Vaccine Antigens in both the <i>Chlamydia trachomatis</i> and <i>Chlamydia muridarum</i> Intravaginal Mouse Challenge Models

Identifying relevant animal challenge models adds to the complexity of human vaccine development. Murine challenge models have been the most utilized animal model for Chlamydia trachomatis vaccine development. The question arises as to whether the C. trachomatis or C. muridarum pre-clinical model is optimal. We compared C. muridarum and C. trachomatis intravaginal challenge models in a combined total of seventy-five studies evaluating potential vaccine candidates. In 100% (42/42) of C. muridarum studies, mice immunized with Chlamydia elementary bodies (EB) demonstrated a significant reduction in urogenital bacterial shedding as measured by qPCR (p C. trachomatis studies. We have evaluated proposed vaccine antigens in both models and observed immunization with Chlamydia major outer membrane protein (MOMP) vaccine formulations to be protective (p C. trachomatis model, and immunization with PmpD p82 translocator domain was not protective in either model. We also observed in both models that depletion of CD4+ T-cells in MOMP-immunized mice resulted in diminished protective immunity but animals were still able to reduce the infection level. In contrast, mice immunized with live EBs by intraperitoneal route did not require CD4+ T-cells to resolve urogenital infection from intravaginal challenge in either model. Overall, we have found the C. muridarum model to be a more robust, reliable, and reproducible model for vaccine antigen discovery.

Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

On mechanisms of colicin import: the outer membrane quandary.

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10-9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic β-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.

Variations in the Botulinum Neurotoxin Binding Domain and the Potential for Novel Therapeutics.

Botulinum neurotoxins (BoNTs) are categorised into immunologically distinct serotypes BoNT/A to /G). Each serotype can also be further divided into subtypes based on differences in amino acid sequence. BoNTs are ~150 kDa proteins comprised of three major functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (HN), and a binding domain (HC). The HC is responsible for targeting the BoNT to the neuronal cell membrane, and each serotype has evolved to bind via different mechanisms to different target receptors. Most structural characterisations to date have focussed on the first identified subtype within each serotype (e.g., BoNT/A1). Subtype differences within BoNT serotypes can affect intoxication, displaying different botulism symptoms in vivo, and less emphasis has been placed on investigating these variants. This review outlines the receptors for each BoNT serotype and describes the basis for the highly specific targeting of neuronal cell membranes. Understanding receptor binding is of vital importance, not only for the generation of novel therapeutics but also for understanding how best to protect from intoxication.

A potential gain-of-function variant of SLC9A6 leads to endosomal alkalinization and neuronal atrophy associated with Christianson Syndrome

Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, complex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R-containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom-shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures.