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BioRxiv : the Preprint Server For... Mar 2024Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate...
UNLABELLED
Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.
SIGNIFICANCE STATEMENT
The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.
PubMed: 38562762
DOI: 10.1101/2024.03.21.585822 -
Biomedical Journal Feb 2024NLRX1 is a member of the of the Nod-like receptor (NLR) family, and it represents a unique pattern recognition molecule (PRM) as it localizes to the mitochondrial matrix... (Review)
Review
NLRX1 is a member of the of the Nod-like receptor (NLR) family, and it represents a unique pattern recognition molecule (PRM) as it localizes to the mitochondrial matrix in resting conditions. Over the past fifteen years, NLRX1 has been proposed to regulate multiple cellular processes, including antiviral immunity, apoptosis, reactive oxygen species (ROS) generation and mitochondrial metabolism. Similarly, in vivo models have shown that NLRX1 was associated with the control of a number of diseases, including multiple sclerosis, colorectal cancer and ischemia-reperfusion injury. This apparent versatility in function hinted that a common and general overarching role for NLRX1 may exist. Recent evidence has suggested that NLRX1 controls mitophagy through the detection of a specific "danger signal", namely the defective import of proteins into mitochondria, or mitochondrial protein import stress (MPIS). In this review article, we propose that mitophagy regulation may represent the overarching process detected by NLRX1, which could in turn impact on a number of diseases if dysfunctional.
Topics: Humans; Mitophagy; Mitochondrial Proteins; NLR Proteins; Mitochondria; Apoptosis
PubMed: 37574163
DOI: 10.1016/j.bj.2023.100635 -
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 -
Science Advances Oct 2023The clinical potential of miRNA-based liquid biopsy has been largely limited by the heterogeneous sources in plasma and tedious assay processes. Here, we develop a...
The clinical potential of miRNA-based liquid biopsy has been largely limited by the heterogeneous sources in plasma and tedious assay processes. Here, we develop a precise and robust one-pot assay called dual-surface-protein-guided orthogonal recognition of tumor-derived exosomes and in situ profiling of microRNAs (SORTER) to detect tumor-derived exosomal miRNAs and enhance the diagnostic accuracy of prostate cancer (PCa). The SORTER uses two allosteric aptamers against exosomal marker CD63 and tumor marker EpCAM to create an orthogonal labeling barcode and achieve selective sorting of tumor-specific exosome subtypes. Furthermore, the labeled barcode on tumor-derived exosomes initiated targeted membrane fusion with liposome probes to import miRNA detection reagents, enabling in situ sensitive profiling of tumor-derived exosomal miRNAs. With a signature of six miRNAs, SORTER differentiated PCa and benign prostatic hyperplasia with an accuracy of 100%. Notably, the diagnostic accuracy reached 90.6% in the classification of metastatic and nonmetastatic PCa. We envision that the SORTER will promote the clinical adaptability of miRNA-based liquid biopsy.
Topics: Male; Humans; Exosomes; Membrane Proteins; MicroRNAs; Prostatic Neoplasms; Biomarkers, Tumor
PubMed: 37792944
DOI: 10.1126/sciadv.adi1556 -
Journal of Cell Science Sep 2023Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in...
Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.
Topics: Ubiquitin; Chloroplasts; Photosynthesis; Proteasome Endopeptidase Complex; Chloroplast Proteins; Protein Transport
PubMed: 37732520
DOI: 10.1242/jcs.241125 -
Trends in Cell Biology May 2024Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even... (Review)
Review
Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even import folded proteins, but the underlying mechanism has been a mystery. Recent results reveal how import receptors shuttle cargo into peroxisomes. The cargo-bound receptors move from the cytosol across the peroxisomal membrane completely into the matrix by a mechanism that resembles transport through the nuclear pore. The receptors then return to the cytosol through a separate retrotranslocation channel, leaving the cargo inside the organelle. This cycle concentrates imported proteins within peroxisomes, and the energy for cargo import is supplied by receptor export. Peroxisomal protein import thus fundamentally differs from other previously known mechanisms for translocating proteins across membranes.
Topics: Peroxisomes; Protein Transport; Humans; Animals; Cytosol; Receptors, Cytoplasmic and Nuclear
PubMed: 37743160
DOI: 10.1016/j.tcb.2023.08.005 -
BioRxiv : the Preprint Server For... Dec 2023The efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A is primarily...
The efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A is primarily known as an elongation factor which binds ribosomes to alleviate ribosome stalling at sequences encoding polyprolines or combinations of proline with glycine and charged amino acids. eIF5A is known to impact the mitochondrial function across a variety of species although the precise molecular mechanism underlying this impact remains unclear. We found that depletion of eIF5A in yeast drives reduced translation and levels of TCA cycle and oxidative phosphorylation proteins. We further found that loss of eIF5A leads to the accumulation of mitoprotein precursors in the cytosol as well as to the induction of a mitochondrial import stress response. Here we identify an essential polyproline-containing protein as a direct eIF5A target for translation: the mitochondrial inner membrane protein Tim50, which is the receptor subunit of the TIM23 translocase complex. We show how eIF5A directly controls mitochondrial protein import through the alleviation of ribosome stalling along mRNA at the mitochondrial surface. Removal of the polyprolines from Tim50 rescues the mitochondrial import stress response, as well as the translation of oxidative phosphorylation reporter genes in an eIF5A loss of function. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by reducing ribosome stalling and facilitating protein translation, thereby positively impacting the mitochondrial import process.
PubMed: 38187585
DOI: 10.1101/2023.12.19.572290 -
Proceedings of the National Academy of... May 2024Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate...
Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.
Topics: Viral Proteins; Bacteriophages; Protein Transport; Cell Nucleus; Virus Replication
PubMed: 38687783
DOI: 10.1073/pnas.2321190121 -
Liver International : Official Journal... Aug 2023Steatosis is the early pathological change in alcohol-associated liver disease. However, its precise mechanism is still unclear. The present study is aimed to explore...
BACKGROUND AND AIMS
Steatosis is the early pathological change in alcohol-associated liver disease. However, its precise mechanism is still unclear. The present study is aimed to explore the role and mechanism of acetyl-CoA synthetase 2 (ACSS2) in acute alcohol-induced lipogenesis.
METHODS
The increase in ACSS2 nuclear import and histone H3 acetylation were observed in mice after intraperitoneally injected with 2 g/kg ethanol or oral administration of 5 g/kg ethanol and also validated in hepatocytes stimulated with ethanol or acetate. The role of ACSS2 was further explored in liver-specific ACSS2 knockdown mice fed with ethanol-containing diet.
RESULTS
Alcohol challenge induced hepatic lipid deposition and upregulated lipogenic genes in mice. It also promoted ACSS2 nuclear import and increased histone H3 acetylation. In hepatocytes, ethanol induced similar phenomena whereas ACSS2 knockdown blocked histone acetylation and lipogenic gene induction. P300/CBP associated factor (PCAF), but not general control nonderepressible 5, CREB-binding protein (CBP) and p300, facilitated H3K9 acetylation responding to ethanol challenge. CUT&RUN assay showed the enrichment of acetylated histone H3K9 surrounding Fasn and Acaca promoters. These results indicated that ethanol metabolism promoted ACSS2 nuclear import to support lipogenesis via H3K9 acetylation. In alcohol-feeding mice, liver-specific ACSS2 knockdown blocked the interaction between PCAF and H3K9 and suppressed lipogenic gene induction in the liver, demonstrating the critical role of ACSS2 in lipogenesis.
CONCLUSIONS
Our study demonstrated that alcohol metabolism generated acetyl-CoA in the nucleus dependently on nuclear ACSS2, contributing to epigenetic regulation of lipogenesis in hepatic steatosis. Targeting ACSS2 may be a potential therapeutical strategy for acute alcoholic liver steatosis.
Topics: Animals; Mice; Acetyl Coenzyme A; Epigenesis, Genetic; Ethanol; Fatty Liver; Fatty Liver, Alcoholic; Histones; Lipogenesis; Liver; Liver Diseases, Alcoholic; Acetate-CoA Ligase
PubMed: 37183518
DOI: 10.1111/liv.15600 -
Nature Cell Biology Apr 2024β-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain...
β-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain iron accumulation. Mutations in the WD repeat domain 45 (WDR45) gene encoding WIPI4 lead to loss of function in BPAN but the cellular mechanisms of how these trigger pathology are unclear. The prevailing view in the literature is that BPAN is simply the consequence of autophagy deficiency given that WIPI4 functions in this degradation pathway. However, our data indicate that WIPI4 depletion causes ferroptosis-a type of cell death induced by lipid peroxidation-via an autophagy-independent mechanism, as demonstrated both in cell culture and in zebrafish. WIPI4 depletion increases ATG2A localization at endoplasmic reticulum-mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria. This results in increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation, thus enabling ferroptosis. This mechanism has minimal overlap with classical ferroptosis stimuli but provides insights into the causes of neurodegeneration in BPAN and may provide clues for therapeutic strategies.
Topics: Animals; Ferroptosis; Zebrafish; Carrier Proteins; Autophagy; Mutation
PubMed: 38454050
DOI: 10.1038/s41556-024-01373-3