Protein Transport Research Articles

BmATAD3A mediates mitochondrial ribosomal protein expression to maintain the mitochondrial energy metabolism of the silkworm, Bombyx mori.

ATAD3A is a mitochondrial membrane protein belonging to the ATPase family that contains the AAA+ domain. It is widely involved in mitochondrial metabolism, protein transport, cell growth, development and other important life processes. It has previously been reported that the deletion of ATAD3A causes growth and development defects in humans, mice and Caenorhabditis elegans. To delve into the mechanism underlying ATAD3A defects and their impact on development, we constructed a Bombyx mori ATAD3A (BmATAD3A) defect model in silkworm larvae. We aim to offer a reference for understanding ATAD3A genetic defects and elucidating the molecular regulatory mechanisms. The results showed that knockout of the BmATAD3A gene significantly affected the weight, survival rate, ATPase production and mitochondrial metabolism of individuals after 24h of incubation. Combined metabolomics and transcriptomics analysis further demonstrated that BmATAD3A knockout inhibits amino acid biosynthesis through the regulation of mitochondrial ribosomal protein expression. Simultaneously, our findings indicate that BmATAD3A knockout impeded mitochondrial activity and ATPase synthesis and suppressed the mitochondrial oxidative phosphorylation pathway through B.mori mitochondrial ribosomal protein L11 (BmmRpL11). These results provide novel insights into the molecular mechanisms involved in the inhibition of development caused by ATAD3A deficiency, offering a potential direction for targeted therapy in diseases associated with abnormal ATAD3A expression.

The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

Biomaterials for Targeting Endoplasmic Reticulum in Cancer.

Endoplasmic reticulum (ER) is one of the most important sub-cellular organelles which controls myriads of biological functions including protein biosynthesis with proper functional folded form, protein misfolding, protein transport into Golgi body for secretion, Ca2+ homeostasis and so on. Subsequently, dysregulation in ER function leads to ER stress followed by disease pathology like cancer. Hence, targeting ER in the cancer cells emerged as one of the futuristic strategies for cancer treatment. However, the major challenge is to selectively and specifically target ER in the sub-cellular milieu in the cancer tissues, due to the lack of ER targeting chemical moieties to recognize the ER markers. To address this, in last decade, numerous biomaterials were explored to selectively impair and image ER in cancer cells to induce ER stress. This review outlines those biomaterials which consists of carbon and silicon materials, lipid nanoparticles (liposomes and micelles), supramolecular self-assembled nanostructures, cell membrane coated nanoparticles and metallic nanoparticles. Moreover, we also discussed the challenges and possible solutions of this promising field to usher the readers towards next-generation ER targeted cancer therapy.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in Saccharomyces cerevisiae

The Sit4 protein phosphatase plays a key role in orchestrating various cellular processes essential for maintaining cell viability during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression, and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, SIT4 exhibited a negative genetic interaction with VPS27, as sit4∆vps27∆ double mutants had a shortened lifespan compared to sit4∆ and vps27∆ single mutants. Our results also show that Vps27 did not increase sit4∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4∆ cells, as sit4∆vps27∆ double mutants exhibited high iron levels and impaired mitochondrial respiration. These findings show, for the first time, cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Allelic heterogeneity and abnormal vesicle recycling in PLAA-related neurodevelopmental disorders

The human PLAA gene encodes Phospholipase-A2-Activating-Protein (PLAA) involved in trafficking of membrane proteins. Through its PUL domain (PLAP, Ufd3p, and Lub1p), PLAA interacts with p97/VCP modulating synaptic vesicles recycling. Although few families carrying biallelic PLAA variants were reported with progressive neurodegeneration, consequences of monoallelic PLAA variants have not been elucidated. Using exome or genome sequencing we identified PLAA de-novo missense variants, affecting conserved residues within the PUL domain, in children affected with neurodevelopmental disorders (NDDs), including psychomotor regression, intellectual disability (ID) and autism spectrum disorders (ASDs). Computational and in-vitro studies of the identified variants revealed abnormal chain arrangements at C-terminal and reduced PLAA-p97/VCP interaction, respectively. These findings expand both allelic and phenotypic heterogeneity associated to PLAA-related neurological disorders, highlighting perturbed vesicle recycling as a potential disease mechanism in NDDs due to genetic defects of PLAA.

Massive ER protein disposal by reticulophagy receptors and selective disposal by TOLLIP

ABSTRACT Proteostasis of the endoplasmic reticulum (ER) is maintained by coordinated action of two major catabolic pathways: proteasome-dependent ER-associated degradation (ERAD) and less characterized lysosomal pathways. Recent studies on ER-specific autophagy (termed “reticulophagy”) have highlighted the importance of lysosomes for ER proteostasis. Key to this process are proteins termed reticulophagy receptors that connect ER fragments and Atg8-family proteins, facilitating the lysosomal degradation of both native and aberrant ER proteins in a relatively nonselective manner. In contrast, our recent work identified TOLLIP as a novel type of cargo receptor specifically dedicated to the lysosomal degradation of aberrant ER membrane proteins. The clients of TOLLIP include an engineered model substrate, which mimics an ER-retained aberrant membrane protein, and motor neuron disease-linked misfolded mutants of VAPB and BSCL2/Seipin. TOLLIP acts as a receptor to connect these aberrant ER membrane proteins and phosphatidylinositol-3-phosphate (PtdIns3P) by recognizing the former through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain, and the latter through its C2 domain. These interactions enable PtdIns3P-dependent vesicular trafficking of aberrant membrane proteins to lysosomes without promoting reticulophagic turnover of bulk ER.

DeepLoc 2.1: multi-label membrane protein type prediction using protein language models.

DeepLoc 2.0 is a popular web server for the prediction of protein subcellular localization and sorting signals. Here, we introduce DeepLoc 2.1, which additionally classifies the input proteins into the membrane protein types Transmembrane, Peripheral, Lipid-anchoredand Soluble. Leveraging pre-trained transformer-based protein language models, the server utilizes a three-stage architecture for sequence-based, multi-label predictions. Comparative evaluations with other established tools on a test set of 4933 eukaryotic protein sequences, constructed following stringent homology partitioning, demonstrate state-of-the-art performance. Notably, DeepLoc 2.1 outperforms existing models, with the larger ProtT5 model exhibiting a marginal advantage over the ESM-1B model. The web server is available at https://services.healthtech.dtu.dk/services/DeepLoc-2.1.

Early Chronic Fluoxetine Treatment of Ts65Dn Mice Rescues Synaptic Vesicular Deficits and Prevents Aberrant Proteomic Alterations

Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS.

Identification of Growth-Related Gene BAMBI and Analysis of Gene Structure and Function in the Pacific White Shrimp Litopenaeus vannamei.

As one of the most important aquaculture species in the world, the improvement of growth traits of the Pacific white shrimp (Litopenaeus vannamei), has always been a primary focus. In this study, we conducted SNP-specific locus analysis and identified a growth-related gene, BAMBI, in L. vannamei. We analyzed the structure and function of LvBAMBI using genomic, transcriptomic, metabolomic, and RNA interference (RNAi) assays. The LvBAMBI possessed highly conserved structural domains and widely expressed in various tissues. Knockdown of LvBAMBI significantly inhibited the gain of body length and weight of the shrimp, underscoring its role as a growth-promoting factor. Specifically, knockdown of LvBAMBI resulted in a significant downregulation of genes involved in lipid metabolism, protein synthesis, catabolism and transport, and immunity. Conversely, genes related to glucose metabolism exhibited significant upregulations. Analysis of differential metabolites (DMs) in metabolomics further revealed that LvBAMBI knockdown may primarily affect shrimp growth by regulating biological processes related to lipid and glucose metabolism. These results suggested that LvBAMBI plays a crucial role in regulating lipid metabolism, glucose metabolism, and protein transport in shrimp. This study provides valuable insights for future research and utilization of BAMBI genes in shrimp and crustaceans.

Systematic investigation of the trafficking of glycoproteins on the cell surface

Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.

EH domain-containing protein 2 (EHD2): Overview, biological function, and therapeutic potential.

EH domain-containing protein 2 (EHD2) is a member of the EHD protein family and is mainly located in the plasma membrane, but can also be found in the cytoplasm and endosomes. EHD2 is also a nuclear-cytoplasmic shuttle protein. After entering the cell nuclear, EHD2 acts as a corepressor of transcription to inhibit gene transcription. EHD2 regulates a series of biological processes. As a key regulator of endocytic transport, EHD2 is involved in the formation and maintenance of endosomal tubules and vesicles, which are critical for the intracellular transport of proteins and other substances. The N-terminal of EHD2 is attached to the cell membrane, while its C-terminal binds to the actin-binding protein. After binding, EHD2 connects with the actin cytoskeleton, forming the curvature of the membrane and promoting cell endocytosis. EHD2 is also associated with membrane protein trafficking and receptor signaling, as well as in glucose metabolism and lipid metabolism. In this review, we highlight the recent advances in the function of EHD2 in various cellular processes and its potential implications in human diseases such as cancer and metabolic disease. We also discussed the prospects for the future of EHD2. EHD2 has a broad prospect as a therapeutic target for a variety of diseases. Further research is needed to explore its mechanism, which could pave the way for the development of targeted treatments.

Is STARD3 A New Biomarker for Breast Cancer?

Is STARD3 A New Biomarker for Breast Cancer?

Placental ontogeny in the Yucca Night Lizard, Xantusia vigilis.

Squamate placentas support physiological exchange between mothers and embryos. Uterine and embryonic epithelial cells provide sites for transporting mechanisms and extraembryonic membranes provide the scaffolding for embryonic epithelial cells and vascular systems. Diversity in placental structure involves variation in extraembryonic membrane development as well as epithelial cell specializations. Variation in placental ontogeny is known to occur and, although lineage specific patterns have been described, phylogenetic distribution of specific patterns is poorly understood. Xantusia vigilis is a viviparous lizard in a monophyletic clade, Xantusiidae, of predominantly viviparous species. Xantusiidae is one of two viviparous lineages within the clade Scincoidea that provides an important outgroup comparison for Scincidae, which includes the largest number of independent origins of viviparity among Squamata. Previous reports contain brief descriptions of placental structure of X vigilis but the developmental pattern is unknown including relevant details for comparison with skinks. We studied placental ontogeny in X. vigilis to address two hypotheses: (1) the pattern of development of placental architecture is similar to species of Scincidae and, (2) placental epithelial cell specializations are similar to species of Scincidae. The terminal placental stage of X. vigilis is similar to skinks in that it includes a chorioallantoic placenta and an omphaloplacenta. The chorioallantoic placenta is richly vascularized with thin, squamous epithelial cells separating the two vascular systems. This morphology differs from the elaborate epithelial cell specializations as occur in some skink species, but is similar to many species. Epithelial cells of the omphaloplacenta are enlarged, as they are in scincids, yet development of the omphaloplacenta includes a vascular pattern known to occur only in gerrhonotine lizards. Histochemical staining properties of the epithelium of the omphalopleure of the omphaloplacenta indicate the potential for protein transport, a function not previously reported for lizards.

Effects of schisandrin B on hypoxia-related cognitive function and protein expression in vascular dementia rats

ABSTRACT Vascular dementia (VD) a heterogenous group of brain disorders in which cognitive impairment is attributable to vascular risk factors and cerebrovascular disease. A common phenomenon in VD is a dysfunctional cerebral regulatory mechanism associated with insufficient cerebral blood flow, ischemia and hypoxia. Under hypoxic conditions oxygen supply to the brain results in neuronal death leading to neurodegenerative diseases including Alzheimer’s (AD) and VD. In conditions of hypoxia and low oxygen perfusion, expression of hypoxia-inducible factor 1 alpha (HIF-1α) increases under conditions of low oxygen and low perfusion associated with upregulation of expression of hypoxia-upregulated mitochondrial movement regulator (HUMMR), which promotes anterograde mitochondrial transport by binding with trafficking protein kinesin 2 (TRAK2). Schisandrin B (Sch B) an active component derived from Chinese herb Wuweizi prevented β-amyloid protein induced morphological alterations and cell death using a SH-SY5Y neuronal cells considered an AD model. It was thus of interest to determine whether Sch B might also alleviate VD using a rat bilateral common carotid artery occlusion (BCAO) dementia model. The aim of this study was to examine the effects of Sch B in BCAO on cognitive functions such as Morris water maze test and underlying mechanisms involving expression of HIF-1α, TRAK2, and HUMMR levels. The results showed that Sch B improved learning and memory function of rats with VD and exerted a protective effect on the hippocampus by inhibition of protein expression of HIF-1α, TRAK2, and HUMMR factors. Evidence indicates that Sch B may be considered as an alternative in VD treatment.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

An Ultra-Rare Mixed Phenotype with Combined AP-4 and ERF Mutations: The First Report in a Pediatric Patient and a Literature Review

The adaptor protein 4 (AP-4) constitutes a conserved hetero-tetrameric complex within the family of adaptor protein (AP) complex, crucial for the signal-mediated trafficking of integral membrane proteins. Mutations affecting all subunits of the AP-4 complex have been linked to autosomal-recessive cerebral palsy and a complex hereditary spastic paraparesis (HSP) phenotype. Our report details the case of a 14-year-old boy born to consanguineous parents, presenting psychomotor delay, severe intellectual disability, microcephaly, and trigonocephaly. Despite a history of febrile seizures, subsequent years were devoid of seizures, with normal EEG. Exome sequencing revealed pathogenic variants in both the AP4B1 and ERF genes. Significantly, the patient exhibited features associated with AP4B1 mutations, including distinctive traits such as cranial malformations. The ERF gene variant, linked to craniosynostosis, likely contributes to the observed trigonocephaly. This case represents the initial documentation of a concurrent mutation in the AP4B1 and ERF genes, underscoring the critical role of exome analysis in unraveling complex phenotypes. Understanding these complex genotypes offers valuable insights into broader syndromic conditions, facilitating comprehensive patient management.

RNAi-mediated knockdown of exportin 1 negatively affected ovary development, survival and maize mosaic virus accumulation in its insect vector Peregrinus maidis.

Exportin 1 (XPO1) is the major karyopherin-β nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.