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Nature Communications Nov 2023Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss...
Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.
Topics: Humans; Ubiquitination; Ubiquitin; Protein Transport; Peroxisomes; Intracellular Membranes
PubMed: 37963875
DOI: 10.1038/s41467-023-43262-7 -
Philosophical Transactions of the Royal... Apr 2024The () gene encodes a core component of the retromer complex essential for the endosomal sorting and recycling of transmembrane cargo. Endo-lysosomal pathway deficits... (Review)
Review
The () gene encodes a core component of the retromer complex essential for the endosomal sorting and recycling of transmembrane cargo. Endo-lysosomal pathway deficits are suggested to play a role in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Mutations in cause a late-onset, autosomal dominant form of PD, with a single missense mutation (D620N) shown to segregate with disease in PD families. Understanding how the PD-linked D620N mutation causes retromer dysfunction will provide valuable insight into the pathophysiology of PD and may advance the identification of therapeutics. D620N VPS35 can induce LRRK2 hyperactivation and impair endosomal recruitment of the WASH complex but is also linked to mitochondrial and autophagy-lysosomal pathway dysfunction and altered neurotransmitter receptor transport. The clinical similarities between -linked PD and sporadic PD suggest that defects observed in cellular and animal models with the mutation may provide valuable insights into sporadic disease. In this review, we highlight the current knowledge surrounding VPS35 and its role in retromer dysfunction in PD. We provide a critical discussion of the mechanisms implicated in -mediated neurodegeneration in PD, as well as the interplay between VPS35 and other PD-linked gene products. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.
Topics: Animals; Humans; Parkinson Disease; Vesicular Transport Proteins; Protein Transport; Neurodegenerative Diseases; Mutation
PubMed: 38368930
DOI: 10.1098/rstb.2022.0384 -
Nucleus (Austin, Tex.) Dec 2024The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the... (Review)
Review
The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the NE is directly continuous with the endoplasmic reticulum (ER), the NE has an overall distinct protein composition from the ER, which is crucial for its functions. During open mitosis in higher eukaryotes, the NE disassembles during mitotic entry and then reforms as a functional territory at the end of mitosis to reestablish nucleocytoplasmic compartmentalization. In this review, we examine the known mechanisms by which the functional NE reconstitutes from the mitotic ER in the continuous ER-NE endomembrane system during open mitosis. Furthermore, based on recent findings indicating that the NE possesses unique lipid metabolism and quality control mechanisms distinct from those of the ER, we explore the maintenance of NE identity and homeostasis during interphase. We also highlight the potential significance of membrane junctions between the ER and NE.
Topics: Nuclear Envelope; Nuclear Pore; Endoplasmic Reticulum; Mitosis; Active Transport, Cell Nucleus
PubMed: 38238284
DOI: 10.1080/19491034.2023.2299632 -
Current Opinion in Cell Biology Dec 2023Regulated secretion, an essential cellular process, relies on secretory granules (SGs) for the controlled release of a diverse range of cargo molecules, including... (Review)
Review
Regulated secretion, an essential cellular process, relies on secretory granules (SGs) for the controlled release of a diverse range of cargo molecules, including proteins, peptides, hormones, enzymes, and neurotransmitters. SG biogenesis encompasses cargo selection, sorting, packaging, and trafficking, with the trans-Golgi Network (TGN) playing a central role. Research in the last three decades has revealed significant components required for SG biogenesis; however, no cargo receptor transferring granule cargo from the TGN to immature SGs (ISGs) has yet been identified. Consequently, recent research has devoted significant attention to studying receptor-independent cargo sorting mechanisms, shedding new light on the complexities of regulated secretion. Understanding the underlying molecular and biophysical mechanisms behind cargo sorting into ISGs holds great promise for advancing our knowledge of cellular communication and disease mechanisms.
Topics: trans-Golgi Network; Proteins; Protein Transport; Biological Transport; Secretory Vesicles
PubMed: 37657367
DOI: 10.1016/j.ceb.2023.102231 -
The Journal of Cell Biology Feb 2024Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C....
Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C. elegans-polarized intestine epithelia, we recently have revealed that the small GTPase RAB-8/Rab8 serves as an important player in the process. Nonetheless, its function and the relevant UcPS itinerary remain poorly understood. Here, we show that deregulated RAB-8 activity resulted in impaired apical UcPS, which increased sensitivity to infection and environmental stress. We also identified the SNARE VTI-1/Vti1a/b as a new RAB-8-interacting factor involved in the apical UcPS. Besides, RAB-11/Rab11 was capable of recruiting RABI-8/Rabin8 to reduce the guanine nucleotide exchange activity of SMGL-1/GEF toward RAB-8, indicating the necessity of a finely tuned RAB-8/RAB-11 network. Populations of RAB-8- and RAB-11-positive endosomal structures containing the apical UcPS cargo moved toward the apical side. In the absence of RAB-11 or its effectors, the cargo was retained in RAB-8- and RAB-11-positive endosomes, respectively, suggesting that these endosomes are utilized as intermediate carriers for the UcPS.
Topics: Animals; Caenorhabditis elegans; Cell Membrane; Protein Transport; Caenorhabditis elegans Proteins; rab GTP-Binding Proteins; Endosomes
PubMed: 38019180
DOI: 10.1083/jcb.202306107 -
Journal of the American Chemical Society Dec 2023The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better...
The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CA), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.
Topics: Humans; Nuclear Proteins; Cell Nucleus; Neurodegenerative Diseases; Transcription Factors; Active Transport, Cell Nucleus; Cell Cycle Proteins
PubMed: 37992275
DOI: 10.1021/jacs.3c06179 -
BMC Bioinformatics Nov 2023Determining a protein's quaternary state, i.e. the number of monomers in a functional unit, is a critical step in protein characterization. Many proteins form multimers...
BACKGROUND
Determining a protein's quaternary state, i.e. the number of monomers in a functional unit, is a critical step in protein characterization. Many proteins form multimers for their activity, and over 50% are estimated to naturally form homomultimers. Experimental quaternary state determination can be challenging and require extensive work. To complement these efforts, a number of computational tools have been developed for quaternary state prediction, often utilizing experimentally validated structural information. Recently, dramatic advances have been made in the field of deep learning for predicting protein structure and other characteristics. Protein language models, such as ESM-2, that apply computational natural-language models to proteins successfully capture secondary structure, protein cell localization and other characteristics, from a single sequence. Here we hypothesize that information about the protein quaternary state may be contained within protein sequences as well, allowing us to benefit from these novel approaches in the context of quaternary state prediction.
RESULTS
We generated ESM-2 embeddings for a large dataset of proteins with quaternary state labels from the curated QSbio dataset. We trained a model for quaternary state classification and assessed it on a non-overlapping set of distinct folds (ECOD family level). Our model, named QUEEN (QUaternary state prediction using dEEp learNing), performs worse than approaches that include information from solved crystal structures. However, it successfully learned to distinguish multimers from monomers, and predicts the specific quaternary state with moderate success, better than simple sequence similarity-based annotation transfer. Our results demonstrate that complex, quaternary state related information is included in such embeddings.
CONCLUSIONS
QUEEN is the first to investigate the power of embeddings for the prediction of the quaternary state of proteins. As such, it lays out strengths as well as limitations of a sequence-based protein language model approach, compared to structure-based approaches. Since it does not require any structural information and is fast, we anticipate that it will be of wide use both for in-depth investigation of specific systems, as well as for studies of large sets of protein sequences. A simple colab implementation is available at: https://colab.
RESEARCH
google.com/github/Furman-Lab/QUEEN/blob/main/QUEEN_prediction_notebook.ipynb .
Topics: Proteins; Amino Acid Sequence; Language; Protein Structure, Secondary; Protein Transport
PubMed: 37964216
DOI: 10.1186/s12859-023-05549-w -
Journal of Neurochemistry Feb 2024The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults,...
The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.
Topics: Animals; Humans; HEK293 Cells; Protein Transport; Endosomes; Endocytosis; Astrocytes; Aquaporin 4; Mammals
PubMed: 38102893
DOI: 10.1111/jnc.16029 -
Biophysical Journal May 2024Cell membranes act as semi-permeable barriers, often restricting the entry of large or hydrophilic molecules. Nonetheless, certain amphiphilic molecules, such as...
Cell membranes act as semi-permeable barriers, often restricting the entry of large or hydrophilic molecules. Nonetheless, certain amphiphilic molecules, such as antimicrobial and cell-penetrating peptides, can cross these barriers. In this study, we demonstrate that specific properties of transmembrane proteins/peptides can enhance membrane permeation of amphiphilic peptides. Using coarse-grained molecular dynamics with free-energy calculations, we identify key translocation-enhancing attributes of transmembrane proteins/peptides: a continuous hydrophilic patch, charged residues preferably in the membrane center, and aromatic hydrophobic residues. By employing both coarse-grained and atomistic simulations, complemented by experimental validation, we show that these properties not only enhance peptide translocation but also speed up lipid flip-flop. The enhanced flip-flop reinforces the idea that proteins such as scramblases and insertases not only share structural features but also operate through identical biophysical mechanisms enhancing the insertion and translocation of amphiphilic molecules. Our insights offer guidelines for the designing of translocation-enhancing proteins/peptides that could be used in medical and biotechnological applications.
Topics: Molecular Dynamics Simulation; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Protein Transport; Lipid Bilayers; Cell Membrane
PubMed: 38615194
DOI: 10.1016/j.bpj.2024.04.009 -
Cells Dec 2023Explaining changes at the gene level that occur during neurodegeneration in the CA3 area is crucial from the point of view of memory impairment and the development of...
Explaining changes at the gene level that occur during neurodegeneration in the CA3 area is crucial from the point of view of memory impairment and the development of post-ischemic dementia. An ischemic model of Alzheimer's disease was used to evaluate changes in the expression of genes related to amyloid transport in the CA3 region of the hippocampus after 10 min of brain ischemia with survival of 2, 7 and 30 days and 12, 18 and 24 months. The quantitative reverse transcriptase PCR assay revealed that the expression of the and genes involved in amyloid transport was dysregulated from 2 days to 24 months post-ischemia in the CA3 area of the hippocampus. gene expression 2 and 7 days after ischemia was below control values. However, its expression from day 30 to 24 months, survival after an ischemic episode was above control values. gene expression 2 days after ischemia was below control values, reaching a maximum increase 7 and 30 days post-ischemia. Then, after 12, 18 and 24 months, it was again below the control values. The data indicate that in the CA3 area of the hippocampus, an episode of brain ischemia causes the increased expression of the gene for 7-30 days during the acute phase and that of from 1 to 24 months after ischemia during the chronic stage. In other words, in the early post-ischemic stage, the expression of the gene that transport amyloid to the brain increases (7-30 days). Conversely, in the late post-ischemic stage, amyloid scavenging/cleaning gene activity increases, reducing and/or preventing further neuronal damage or facilitating the healing of damaged sites. This is how the new phenomenon of pyramidal neuronal damage in the CA3 area after ischemia is defined. In summary, post-ischemic modification of the and genes is useful in the study of the ischemic pathways and molecular factors involved in the development of Alzheimer's disease.
Topics: Humans; Alzheimer Disease; Amyloidogenic Proteins; Brain Ischemia; Hippocampus; Ischemia; Low Density Lipoprotein Receptor-Related Protein-1; tau Proteins; Protein Transport
PubMed: 38067191
DOI: 10.3390/cells12232763