Intracellular Protein Trafficking Research Articles

MTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

The Novel Long QT Syndrome Type 2-associated F129I Mutation in the KCNH2 Gene Significantly Affects I Kr Through the hERG1 Homomeric and Heteromeric Potassium Channels

Objective: The long QT syndrome type 2 is caused by the loss-of-function mutations in the KCNH2 gene, which encodes hERG1, the voltage-gated potassium channel. The hERG1 channels conduct rapid delayed rectifier K+ currents (I Kr) in the human cardiac tissue. KCNH2 encodes 2 main isoforms—hERG1a and hERG1b, which assemble to form the homomeric or heteromeric hERG1 channels. However, the functional characteristics of the heteromeric hERG1 channels in long QT syndrome type 2 are not clear. In this study, a novel mutation in the N-terminus of hERG1a (F129I) was identified in a proband of long QT syndrome type 2. The purpose of this study was to identify the electrophysiological change of homomeric and heteromeric hERG1 channels with the F129I-hERG1a. Methods: Candidate genes were screened by direct sequencing. F129I-hERG1a was cloned in the pcDNA3.1 vector by site-directed mutagenesis. Then, the wild-type (WT) hERG1a and/or F129I-hERG1a were transiently expressed in the HEK293 cells with or without hERG1b co-expression. The expression levels of the transgenes, cellular distribution of hERG1a and hERG1b, and the electrophysiological features of the homomeric and the heteromeric hERG1 channels with the WT-hERG1a or F129I-hERG1a were analyzed using whole-cell patch-clamp electrophysiology, western blotting, and immunofluorescence techniques. Results: The proband was clinically diagnosed with long QT syndrome type 2 and carried a heterozygous mutation c.385T>A (F129I) in the KCNH2 gene. Electrophysiology study proved that the F129I substitution in hERG1a significantly decreased I Kr in both the homomeric and heteromeric hERG1channels by 86% and 70%, respectively (WT-hERG1a (54.88 ± 18.74) pA/pF vs. F129I-hERG1a (7.34 ± 1.90) pA/pF, P < 0.001; WT-hERG1a/hERG1b (89.92 ± 24.51) pA/pF vs. F129I-hERG1a/hERG1b (26.54 ± 9.83) pA/pF, P < 0.001). The voltage dependence of IKr activation (V½ and k) was not affected by the mutation in both the homomeric and heteromeric hERG1 channels. The peak current densities and the kinetic characteristics of IKr were comparable for both WT/F129I-hERG1a and WT-hERG1a. The channel inactivation and deactivation analysis showed that F129I substitution did not affect deactivation of the homomeric hERG1a channel, but significantly accelerated the deactivation and recovery from inactivation of the heteromeric hERG1a/hERG1b channel based on the time constants of fast and slow recovery from deactivation F129I-hERG1a/hERG1b vs. WT-hERG1a/hERG1b (P < 0.05). Western blotting and immunofluorescence labeling experiments showed that maturation and intracellular trafficking of the F129I-hERG1a protein was impaired and potentially increased the ratio of hERG1b to hERG1a in the F129I-hERG1a/hERG1b tetramer channel, thereby resulting in electrophysiological changes characteristic of the long QT syndrome type 2 pathology. Conclusions: I Kr was significantly reduced in the homomeric and heteromeric hERG1 channels with F129I-hERG1a. The F129I mutation significantly accelerated the deactivation and recovery from inactivation of the heteromeric F129I-hERG1a/hERG1b channel. F129I-hERG1a exhibited impaired maturation and intracellular trafficking, thereby potentially increasing the ratio of the hERG1b to hERG1a stoichiometry in the hERG1 tetrameric channel. These changes demonstrated the importance of the heteromeric hERG1 channel in long QT syndrome type 2 pathophysiology.

Ras-Related Protein Rab24 is a Predictor for Hepatocellular Carcinoma Prognosis and Promotes Tumor Growth

Ras-related protein Rab24, a member of the small GTPase family, plays a vital role in regulating intracellular protein trafficking. Recent research has uncovered dysregulation of Rab24 in hepatocellular carcinoma (HCC), but its clinical implications and tumor-related effects require further investigation. We aimed to investigate Rab24’s expression patterns and its role in HCC progression. We analyzed Rab24 expression in HCC and adjacent tissues at the transcriptional, mRNA, and protein levels. The prognostic significance of Rab24 in HCC was assessed through univariate and multivariate analyses, along with Kaplan–Meier survival analysis. Rab24’s impact on cell proliferation was investigated through cellular and xenograft experiments. Our findings revealed elevated Rab24 expression in HCC tissues compared to adjacent liver tissues. High Rab24 expression correlated with larger tumor size and advanced tumor stage. Additionally, HCC patients with high Rab24 expression experienced poorer overall survival, with Rab24 identified as an independent prognostic factor. Manipulating Rab24 expression in HCC cell lines demonstrated its role in promoting tumor proliferation. Silencing Rab24 significantly reduced xenograft growth in vivo. This study highlights the significant association between high Rab24 expression and poorer HCC prognosis, suggesting Rab24’s potential as a novel clinical biomarker and therapeutic target.

Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.Graphical abstract

AGC kinases OXI1 and AGC2-2 regulate camalexin secretion and disease resistance by phosphorylating transporter PDR6.

Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two Orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, Oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three Pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.

Regulation of cardiomyocyte intracellular trafficking and signal transduction by protein palmitoylation.

Despite the well-established functions of protein palmitoylation in fundamental cellular processes, the roles of this reversible post-translational lipid modification in cardiomyocyte biology remain poorly studied. Palmitoylation is catalyzed by a family of 23 zinc finger and Asp-His-His-Cys domain-containing S-acyltransferases (zDHHC enzymes) and removed by select thioesterases of the lysophospholipase and α/β-hydroxylase domain (ABHD)-containing families of serine hydrolases. Recently, studies utilizing genetic manipulation of zDHHC enzymes in cardiomyocytes have begun to unveil essential functions for these enzymes in regulating cardiac development, homeostasis, and pathogenesis. Palmitoylation co-ordinates cardiac electrophysiology through direct modulation of ion channels and transporters to impact their trafficking or gating properties as well as indirectly through modification of regulators of channels, transporters, and calcium handling machinery. Not surprisingly, palmitoylation has roles in orchestrating the intracellular trafficking of proteins in cardiomyocytes, but also dynamically fine-tunes cardiomyocyte exocytosis and natriuretic peptide secretion. Palmitoylation has emerged as a potent regulator of intracellular signaling in cardiomyocytes, with recent studies uncovering palmitoylation-dependent regulation of small GTPases through direct modification and sarcolemmal targeting of the small GTPases themselves or by modification of regulators of the GTPase cycle. In addition to dynamic control of G protein signaling, cytosolic DNA is sensed and transduced into an inflammatory transcriptional output through palmitoylation-dependent activation of the cGAS-STING pathway, which has been targeted pharmacologically in preclinical models of heart disease. Further research is needed to fully understand the complex regulatory mechanisms governed by protein palmitoylation in cardiomyocytes and potential emerging therapeutic targets.

A286 MYO5B MUTATIONS AS A CAUSE OF CHOLESTASIS IN A CHILD WITHOUT MICROVILLOUS INCLUSION DISEASE

Abstract Background Biallelic Myosin Vb (MYO5B) mutations are identified in the majority of patients with microvillus inclusion disease (MVID) that requires life-long parenteral nutrition and eventually cholestatic liver disease. Biallelic MYO5B mutations have also been reported in a few children with predominant early-onset cholestatic liver disease. Aims To report on a patient with MYO5B mutations and progressive familial intrahepatic cholestasis PFIC-like phenotype and normal serum GGT without intestinal disease. Methods We describe a 2-year and 8-month-old girl, who is the first child of healthy non- consanguineous parents from the Phillipines with no family history of liver disease. She presented at the age of 10 months for evaluation of mild jaundice, which was noticed at the age of 6 months. Other symptoms included pruritus, slowly weight gain, intermittent pale stools and dark urine. Laboratory investigations revealed AST 99, ALT 87, GGT 27, conjugated bilirubin 13 and INR 0.8. Fasting abdominal ultrasound was normal apart from a possibly hypoplastic gallbladder and mild splenomegaly. Additional investigations revealed normal thyroid-stimulating hormone, cortisol, galactosemia screening and sweat chloride, but bile acids were elevated 391.6 umol/L. A liver biopsy was performed and a cholestasis genetic panel sent. The patient was started on ursodiol, hydroxyzine, rifampicin and vitamin D. Results Liver biopsy revealed moderate hepato-canalicular cholestasis, bile duct injury without prominent ductopenia, small caliber bile ducts with no features of obstructive cholangiopathy, mild periportal and pericentral fibrosis stage 1-2/ 4. Electron microscopy demonstrated cytoplasmic and canalicular bile accumulation. The bile canaliculi were variably distended by coarse granular bile. Immunohistochemical staining for BSEP and MDR3, showed canalicular staining in zones 1 and absence of staining in zones 2 and 3. Genetic analysis revealed one pathogenic variant and one variant of uncertain significance in MYO5B. No mutations in ABCB11 or ATP8B1 genes were identified. On follow-up, the jaundice improved, pruritus was managed with the medications described above. Her growth has improved and she is growing on the 25th centiles. She has not developed any gastrointestinal symptoms. Conclusions We present a patient with MYO5B mutations who presented with normal GGT cholestasis and a liver biopsy consistent with PFIC, without any evidence of intestinal disease. Because MYO5B mutations disrupt the intracellular trafficking of brush border proteins in the intestine, it has been assumed that it similarly disrupts the trafficking of the canalicular bile acid transporting and bilirubin transporting proteins in the liver. This case highlights the broad phenotype associated with MYO5B variants. Funding Agencies None

Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90β

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks of vascular disorders and acute infectious diseases and related conditions, including the coronavirus disease 2019 (COVID-19) and sepsis. Ubiquitination plays a critical role in regulating the stability, intracellular trafficking, and enzymatic activity of proteins and is reversed by deubiquitinating enzymes (DUBs). The role of DUBs in endothelial biology is largely unknown. In this study, we report that USP40, a poorly characterized DUB, prevents EC barrier disruption through reductions in the activation of RhoA and phosphorylation of myosin light chain (MLC) and cofilin. Furthermore, USP40 reduces EC inflammation through the attenuation of NF-ĸB activation, ICAM1 expression, and leukocyte-EC adhesion. We further show that USP40 activity and expression are reduced in response to endotoxin challenge. Global depletion of USP40 and EC-targeted USP40 depletion in mice exacerbated experimental lung injury, whereas lentiviral gene transfer of USP40 protected against endotoxin-induced lung injury. Using an unbiased approach, we discovered that the protective effect of USP40 occurs through the targeting of heat shock protein 90β (HSP90β) for its deubiquitination and inactivation. Together, these data reveal a critical protective role of USP40 in vascular injury, identifying a unique mechanistic pathway that profoundly impacts endothelial function via DUBs.

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexan Cryptosporidium parvum.

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.

Characterization of a Novel Mutation in the COPI Vesicle on Binding to Dilysine Motifs

Background/Objective: The heptameric COPI coatomer complex is involved in the formation of vesicles and the intracellular trafficking of proteins between the Golgi and Endoplasmic Reticulum as well as throughout the cytoplasm. Members of the COPI complex bind dilysine motifs found in the C-terminal domain of the cargo protein, particularly KKxx or KxKxx. We generated a point mutation in the WD40 domain of the COPI alpha subunit (⍺-COP). We hypothesized that the E269V mutant ⍺-COP would not co-immunoprecipitate (co-IP) COPI cargo proteins terminating with a dilysine domain of KxKxx (Nucleolin and Stasimon/Tmem41b), but would bind cargo proteins terminating in KKxx (FLAGSyntaxin17). We predicted that a mutation in the ⍺-COP C-terminus, which impairs interaction with ε–COP, would not affect its ability to co-IP dilysine-containing cargo.
 Experimental Design or Project Methods: HEK-293TT cells were transfected with Myctagged wild-type, E269V, and triple mutant (3X) ⍺-COP. The E269V ⍺-COP mutant has an amino acid change at position 269 from glutamic acid to valine. The triple ⍺-COP mutant has three amino acid changes that eliminate binding with the ε–COP COPI subunit. Transfected ⍺-COP was immunoprecipitated using magnetic anti-Myc beads. Endogenous Nucleolin was immunoprecipitated using magnetic Protein A beads conjugated to rabbit polyclonal anti- Nucleolin antibody. Western blots of inputs and immunoprecipitates of each experiment were conducted to determine the ability of ⍺-COP to co-IP C-terminal dilysine-containing proteins.
 Results: Endogenous Nucleolin and Stasimon co-immunoprecipitated with WT and 3X ⍺-COP, but not E269V ⍺-COP.
 Conclusions and Potential Impact: The inability of mutation E269V to co-IP dilysine proteins implies that the WD40 domain of the COPI ⍺-COP protein is required for binding to KxKxxterminating proteins, as typified by Nucleolin and Stasimon.The C-terminal 3X mutation shows that ε-COP is not necessary for dilysine recognition and implies that ⍺-COP directly binds to this KxKxx motif.

Revealing protein trafficking by proximity labeling-based proteomics

Protein trafficking is a fundamental process with profound implications for both intracellular and intercellular functions. Proximity labeling (PL) technology has emerged as a powerful tool for capturing precise snapshots of subcellular proteomes by directing promiscuous enzymes to specific cellular locations. These enzymes generate reactive species that tag endogenous proteins, enabling their identification through mass spectrometry-based proteomics. In this comprehensive review, we delve into recent advancements in PL-based methodologies, placing particular emphasis on the label-and-fractionation approach and TransitID, for mapping proteome trafficking. These methodologies not only facilitate the exploration of dynamic intracellular protein trafficking between organelles but also illuminate the intricate web of intercellular and inter-organ protein communications.

Cognitively Healthy Centenarians are genetically protected against Alzheimer’s disease

AbstractBackgroundThe prevalence of Alzheimer’s Disease (AD) increases with age, yet a small fraction of the population reaches ages beyond 100 years maintaining cognitive abilities, so‐called cognitively‐healthy‐centenarians. We aimed to uncover the genetic factors associated with such protection against AD, and to highlight the effects of AD‐associated variants during advanced aging.MethodPrevious Genome‐Wide‐Association‐Studies (GWAS) identified 86 single‐nucleotide‐polymorphisms (SNPs) associated with AD‐risk. We investigated the frequency of each SNP in 346 cognitively‐healthy‐centenarians (mean age 101.05±2.51), compared to 2,281 AD cases (mean age 67.96±9.84), 3,165 middle‐aged healthy controls (mean age 62.57±8.66), and we combined the SNPs into Polygenic Risk Scores (PRS) for each individual. Finally, we characterized the functional properties of the SNPs enriched/depleted the most in the centenarians.ResultAt the SNP level, centenarians were depleted with risk‐increasing SNPs and enriched with protective SNPs. The AD‐related PRS (excluding APOE SNPs) was significantly lower in centenarians; not only compared to AD (OR = 1.96, p = 4.07×10−26), but also to middle‐aged controls (OR = 1.29, p = 2.61×10−5). When including APOE SNPs these effects were even stronger (OR = 5.07, p = 6.15×10−71, and OR = 1.87, p = 4.67×10−17, for comparisons with AD and normal controls, respectively). The most depleted/enriched SNPs in centenarians functionally mapped to activation/regulation of immune response and endocytosis/phagocytosis. These SNPs were located near ANKH, TMEM106B, CLNK, EPDR1, WDR12, PLCG2, RIN3, CD2AP, MS4A, TREM2, ABCA7 and MAF.ConclusionThe ability to reach advanced age without cognitive decline is influenced by AD‐associated genetic variants. An advantageous regulation of the brain’s immune system, intracellular protein trafficking, and endocytosis/phagocytosis may confer the strongest protection against AD and potentially other age‐related diseases.

Key Subdomains of Cerebral Dopamine Neurotrophic Factor Regulate Its Protective Function in 6-Hydroxydopamine-Lesioned PC12 Cells.

Cerebral dopamine neurotrophic factor (CDNF) is a unique neurotrophic factor (NTF) that has shown significant neuroprotective and neurorestorative functions on midbrain dopaminergic neurons. The secondary structure of human CDNF protein contains eight α-helices. We previously found that two key helices, α1 and α7, regulated the intracellular trafficking and secretion of CDNF protein in different manners. The α1 mutation (M1) induced most CDNF proteins to reside in the endoplasmic reticulum and little be secreted extracellularly, while the α7 mutation (M7) caused the majority of CDNF proteins to be secreted out of the cells and little reside in the cells. However, the regulation of the two mutants on the function of CDNF remains unclear. In this study, we investigated the effects of M1 and M7 on the protective activity of CDNF in PC12 cells, which were treated with 6-hydroxydopamine (6-OHDA) to mimic Parkinson's disease. We found that both M1 and M7 could promote survival and inhibit apoptosis more effectively than Wt in 6-OHDA-lesioned PC12 cells. Therefore, these findings will advance our understanding of the important regulation of subdomains on the function of NTFs.

OR14-01 Contact Between Thyroglobulin Opposite Domains Assists Its Upstream Regions In Intracellular Trafficking For Thyroid Hormonogenesis

Abstract Disclosure: C.E. Citterio: None. K. Kim: None. B. Rajesh: None. K. Pena: None. O.B. Clarke: None. P. Arvan: None. Thyroglobulin (Tg), must be released into the lumen of thyroid follicles to facilitate thyroid hormone synthesis. The Tg primary sequence (∼2,760 amino acids) includes regions I, II, III, as well as ∼570 residues of cholinesterase-like (ChEL) domain (which bears 47% similarity to acetylcholinesterase), folded as a globular protein with multiple contacts between different Tg regions. The ChEL domain is reported to function as an intramolecular chaperone assisting Tg region I. This function cannot be replaced by acetylcholinesterase or neuroligins, indicating a unique function of Tg ChEL. However, the specific amino acids of ChEL neighboring upstream Tg regions have only recently been identified, and the key functional residues for Tg folding remain unknown. Here, we’ve combined structural analyses with functional characterization of recombinant Tg expressed in cell culture. We’ve identified hTg-ChEL K2223 and W2222 as amino acids that exhibit the closest physical proximity with Tg region I. By cryo-EM of Tg (both human and bovine), Tg-K2223 and Tg-W2222 reside only ∼4.5Å from Tg-W1089 and Tg-P1098, respectively, both of which are located in the Tg 1-9 repeat within region I. We’ve employed site-directed mutagenesis to determine the impact of these contacts by studying both full length Tg as well as Tg expressed in a two-component system in cultured cells. We find that secretory ChEL-K2223E and ChEL-W2222A behave much like WT secretory ChEL; both mutants are reasonably well expressed and secreted efficiently. However, when secretory ChEL was co-expressed with Tg region I-II-III lacking its own ChEL domain, region I-II-III secretion could be rescued in the presence of WT secretory ChEL, but not by co-expression of either ChEL-K2223E or ChEL-W2222A. In context of a single contiguous full length Tg, both expression and secretion of Tg-K2223E and Tg-W2222A were significantly diminished. These results highlight two residues (K2223 and W2222) that are not needed for the independent folding (or endoplasmic reticulum export) of the Tg ChEL domain, but are key residues for contacting Tg region I, thereby offering the overall Tg structure a great increase in conformational stability, which is needed to satisfy endoplasmic reticulum quality control requirements for intracellular protein trafficking. These findings point to the possibility that mutations altering regional contacts within the Tg structure could contribute to the molecular pathogenesis of congenital hypothyroidism. Presentation: Friday, June 16, 2023

Cell-specific secretory granule sorting mechanisms: the role of MAGEL2 and retromer in hypothalamic regulated secretion.

Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.

SPAG17 mediates nuclear translocation of protamines during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Abstract P2030: Regulation Of Pathologic Cardiac Signaling Via Rac1 Palmitoylation

Development of more effective therapies to prevent maladaptive cardiac remodeling and heart failure would be greatly aided by the identification of novel signaling pathways that regulate cardiac hypertrophy. Cysteine palmitoylation is a reversible post-translational lipid modification that dynamically regulates intracellular trafficking and subcellular compartmentalization of signaling proteins. Previous work from our group identified that mice with cardiomyocyte (CM)-specific overexpression of zDHHC3, a Golgi-localized S-acyltransferase, develop lethal dilated cardiomyopathy. S-acyl-proteomics revealed Cys-178 of Rac1, a small GTPase, as a palmitoylation site modified by zDHHC3 and hearts from zDHHC3-overexpressing mice display elevated levels of palmitoylated, active, and total Rac1, suggesting Rac1 palmitoylation promotes its activation and may drive pathologic cardiac remodeling. Rac1-dependent activation of NADPH oxidase 2 (Nox2) in CMs is required for angiotensin II (AngII)-induced cardiac hypertrophy and myocardial oxidative stress, suggesting inhibition of Rac1/Nox2 signaling could potentially ameliorate adverse cardiac remodeling. We found Rac1 palmitoylation at Cys-178 to be required for hypertrophy and oxidative stress in neonatal rat CMs as reactive oxygen species production and hypertrophy elicited by expression of a constitutively active Rac1 mutant were attenuated by mutation of the Cys-178 palmitoylation site, suggesting palmitoylation targets Rac1 to the Nox2 complex to promote myocardial oxidative stress. Notably, Rac1 palmitoylation and oxidative stress were also enhanced in hearts of mice subjected to AngII infusion and transgenic mice with CM-specific overexpression of the angiotensin II receptor type I (AT1R), suggesting a role for Rac1 palmitoylation in AngII-induced maladaptive cardiac signaling. To further elucidate the role of Rac1 palmitoylation in cardiac pathology, we developed a CM-specific knock-in mouse model in which Cys-178 of Rac1 is mutated to serine. Ongoing work utilizing this model will investigate mechanisms by which palmitoylated and depalmitoylated Rac1 differentially regulate signaling, oxidative stress, and hypertrophy in CMs in response to pathologic stimuli.

AMPK boosts ADAM10 shedding activity in human aortic endothelial cells by promoting Rab14-dependent ADAM10 cell surface translocation

A disintegrin and metalloprotease 10 (ADAM10) regulates the expression of cell surface receptors such as tumor necrosis factor receptor 1, toll-like receptor 4, and the receptor for advanced glycation end products (RAGE) by cleaving their extracellular regions. To function as a sheddase, ADAM10 should translocate from the intracellular compartments to the cell surface, but the translocation mechanism remains unclear. In this study, we explored the possible role of adenosine monophosphate-activated protein kinase (AMPK) in the induction of ADAM10 shedding activity. In cultured human aortic endothelial cells (HAECs), 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator, boosted ADAM10 cell surface translocation and ectodomain shedding of RAGE. ADAM10 inhibition with GI 254023X and ADAM10 siRNA silencing both prevented AICAR-induced RAGE ectodomain shedding. AICAR increased AMPK phosphorylation as well. Both Compound C-mediated AMPK inhibition and AMPKα1-siRNA-mediated AMPK depletion suppressed AICAR-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. On the other hand, siRNA knockdown of Rab14, a small GTPase that facilitates the intracellular trafficking of transmembrane proteins, prevented AICAR-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. In conclusion, AMPK activation is an obvious inducer of ADAM10 shedding activity. Our findings suggest that AMPK boosts ADAM10 shedding activity in HAECs by promoting Rab14-dependent ADAM10 cell surface translocation.

SNARE Protein Snc1 Is Essential for Vesicle Trafficking, Membrane Fusion and Protein Secretion in Fungi.

Fungi are an important group of microorganisms that play crucial roles in a variety of ecological and biotechnological processes. Fungi depend on intracellular protein trafficking, which involves moving proteins from their site of synthesis to the final destination within or outside the cell. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are vital components of vesicle trafficking and membrane fusion, ultimately leading to the release of cargos to the target destination. The v-SNARE (vesicle-associated SNARE) Snc1 is responsible for anterograde and retrograde vesicle trafficking between the plasma membrane (PM) and Golgi. It allows for the fusion of exocytic vesicles to the PM and the subsequent recycling of Golgi-localized proteins back to the Golgi via three distinct and parallel recycling pathways. This recycling process requires several components, including a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), a retromer submit, and the COPI coat complex. Snc1 interacts with exocytic SNAREs (Sso1/2, Sec9) and the exocytic complex to complete the process of exocytosis. It also interacts with endocytic SNAREs (Tlg1 and Tlg2) during endocytic trafficking. Snc1 has been extensively investigated in fungi and has been found to play crucial roles in various aspects of intracellular protein trafficking. When Snc1 is overexpressed alone or in combination with some key secretory components, it results in enhanced protein production. This article will cover the role of Snc1 in the anterograde and retrograde trafficking of fungi and its interactions with other proteins for efficient cellular transportation.