Journal of Biological Chemistry最新文献

Zinc finger antiviral protein (ZAP) binds CpG dinucleotides in viral RNA and targets them for decay. ZAP interacts with several cofactors to form the ZAP antiviral system, including KHNYN, a multidomain endoribonuclease required for ZAP-mediated RNA decay. However, it is unclear how the individual domains in KHNYN contribute to its activity. Here, we demonstrate that the KHNYN amino terminal extended-diKH (ex-diKH) domain is required for antiviral activity and present its crystal structure. The structure belongs to a rare group of KH-containing domains, characterized by a non-canonical arrangement between two type-1 KH modules, with an additional helical bundle. N4BP1 is a KHNYN paralog with an ex-diKH domain that functionally complements the KHNYN ex-diKH domain. Interestingly, the ex-diKH domain structure is present in N4BP1-like proteins in lancelets, which are basal chordates, indicating that it is evolutionarily ancient. While many KH domains demonstrate RNA binding activity, biolayer interferometry and electrophoretic mobility shift assays indicate that the KHNYN ex-diKH domain does not bind RNA. Furthermore, residues required for canonical KH domains to bind RNA are not required for KHNYN antiviral activity. By contrast, an inter-KH domain cleft in KHNYN is a potential protein-protein interaction site and mutations that eliminate arginine salt bridges at the edge of this cleft decrease KHNYN antiviral activity. This suggests that this domain could be a binding site for an unknown KHNYN cofactor.

Viruses transmitted by biting arthropods, arboviruses, pose a significant global health and economic threat. Climate change is exacerbating this issue by expanding the range of disease-carrying vectors. Effective control of arbovirus transmission often relies on targeting the vectors, making it crucial to understand the interactions between the virus and its vector. The exogenous siRNA (exo-siRNA) pathway is a key antiviral defence mechanism in mosquitoes such as Aedes aegypti. Argonaute 2 (Ago2) is a central protein in this pathway, responsible for antiviral activity. While the PIWI domain of Ago proteins is known to mediate slicing activity, not all Ago proteins possess this slicing function. To understand the antiviral mechanism of Ago2 in Ae. aegypti, we aimed to confirm the presence of the catalytic tetrad, a group of amino acids known to be crucial for slicing activity. Here, we confirmed the tetrad (D740, E780, D812, and H950) in Ae. aegypti Ago2 and demonstrated its essential role in antiviral and siRNA pathway activity. Our findings show that the catalytic tetrad is necessary for the degradation of siRNA passenger strands. When the tetrad is absent, siRNA duplexes accumulate, leading to a loss of siRNA pathway function. This underscores the critical role of the tetrad in the antiviral defence mechanism of Ae. aegypti.

Microvillus Inclusion Disease (MVID) is a rare congenital diarrheal disorder typically caused by loss of function mutations in the unconventional myosin, Myosin 5b (MYO5B), which leads to the mis-trafficking of apical components in enterocytes. MVID can manifest in two phenotypes: in both the intestine and liver or the liver alone. Although previous studies seeking to understand MVID disease pathology used MYO5B knockout models, many patients have point mutations and thus express a dysfunctional MYO5B. How these point mutations lead to a broad spectrum of disease severity and the development of two distinct disease phenotypes is still not known. Here, we investigate the effect of MVID patient mutations on the function of the MYO5B motor domain, independent of cargo binding, using confocal imaging and fluorescence recovery after photobleaching. Patient mutations demonstrated a range of effects in these assays, from rigor-like behavior to loss of actin binding. Additionally, analysis of FRAP turnover kinetics suggests that some mutations negatively impact the ability of MYO5B to stay bound to actin. Collectively, our findings indicate that patient mutations affect the MYO5B motor domain in diverse ways, consistent with the spectrum of phenotypes observed in patients.

Alpha-tocopherol (vitamin E) is a plant-derived dietary lipid that is essential for the health of most animals, including humans. Originally discovered as a fertility factor in rodents, the primary health-promoting properties of the vitamin in humans was shown to be protection of neuromuscular functions. Heritable vitamin E deficiency manifests in spinocerebellar ataxia that can be stabilized by timely supplementation with high-dose α-tocopherol. The molecular basis for α-tocopherol's biological activities has been attributed primarily to the vitamin's efficacy in preventing lipid peroxidation in membranes and lipoproteins, but the possibility that the vitamin possesses additional biological activities has been postulated and debated in the literature without conclusive resolution. We designed and synthesized a novel analog of α-tocopherol, 6-hydroxymethyl α-tocopherol (6-HMTC), which retains most of the vitamin's structural, physical and biochemical properties, yet lacks measurable radical-trapping antioxidant activity. 6-HMTC bound to the tocopherol transfer protein with high (nM) affinity, like that of the natural vitamin, attesting to the analog's preservation of structural integrity. Yet, 6-HMTC did not inhibit lipid peroxidation or associated ferroptotic cell death. Notably, 6-HMTC modulated the expression of some genes in a manner essentially identical to that exhibited by α-tocopherol. These findings support the notion that α-tocopherol modulates gene expression via an antioxidant-independent mechanism.

Adaptations to fluctuating environmental conditions require bacteria to make large scale proteomic shifts on short timescales. We previously characterised the tri-partite RimABK protein complex responsible for the post translational modification of the ribosome in response to environmental cues. Regulated control of RpsF polyglutamylation by RimK rapidly influenced the proteome of Pseudomonas fluorescens cells to facilitate colonisation of the plant rhizosphere. Here, we conduct a detailed investigation of the RimB protease. We show RimB to be a bifunctional retropepsin-like aspartic endopeptidase that uniquely recognises and removes glutamate residues from polyglutamated RpsF and stimulates poly-α-L-glutamate synthesis by RimK. We determine the minimal recognition requirements for RimB proteolysis and identify the catalytic aspartate residue required for function. Further, we identify a novel hybrid enzyme composed of RimB and RimK domains that also possesses protease activity. Phylogenetic analysis of accessions encoding either the hybrid or individual RimB and RimK proteins reveals a pattern of rim gene evolution that is distinct from that of the host organisms and reveals potential alternative targets of RimB.

Septins are cytoskeletal filament-forming proteins that typically associate with membranes and perform critical functions in a variety of cellular processes. Septins often colocalize with actin and microtubule structures, yet our understanding of all the ways that septins contribute mechanistically to actin- and microtubule-based functions is incomplete. The Cdc42 effector protein Cdc42EP3 (also known as BORG2) promotes septin localization to actin structures in vivo, but little else is known about how Cdc42EP3 influences the interactions of septins and F-actin. Here, using purified components, we show that Cdc42EP3 binds directly to septins, actin filaments and actin monomers. Moreover, septin-bound Cdc42EP3 accelerates actin filament polymerization. Thus, Cdc42EP3 is not merely a factor that crosslinks septins and F-actin, but one that promotes the formation of actin polymers along septin scaffolds.

Hundreds of thousands die annually from malaria caused by Plasmodium falciparum (Pf), with the emergence of drug-resistant parasites hindering eradication efforts. Antimicrobial peptides (AMPs) are known for their ability to disrupt pathogen membranes without targeting specific receptors, thereby reducing the chance of drug resistance. However, their effectiveness and the biophysical mechanisms by which they target the intracellular parasite remain unexplored. Here, by using native and synthetic AMPs, we discovered a selective mechanism that underlies the anti-malaria activity. Remarkably, the AMPs exclusively interact with Pf-infected Red Blood Cells (Pf-iRBCs), disrupting the cytoskeletal network and reaching the enclosed parasites with correlation to their activity. Moreover, we showed that the unique feature of reduced cholesterol content in the membrane of the infected host makes Pf-iRBCs susceptible to AMPs. Overall, this work highlights the Achilles' heel of malaria parasite and demonstrates the power of AMPs as potential antimalarial drugs with reduced risk of resistance.

Hypoxia and ischemia damage sensitive organelles such as mitochondria, thus mitochondrial dysfunction contributes to metabolic disorders in crustaceans under hypoxia. The mechanisms associated with ferroptosis in hypoxic disorders have not been determined in crustaceans. In particular, the early molecular events of mitochondrial dynamics in crustaceans require clarification. In this study, two evolutionarily conserved mitochondrial fission proteins, Drp1 and MTP18, were identified in the oriental river prawn (Macrobrachium nipponense). In vitro, ferroptosis-mediated impairment of mitochondrial membrane potential was induced by hypoxia in oriental river prawn hemocytes. In hypoxia-induced hemocytes, activation of Drp1 by increased phosphorylation at S616 was identified. Drp1 mitochondrial translocation also increased, and mitochondrial fusion-related protein expression decreased in vivo. Altered mitochondrial fission-fusion dynamics have been linked to mitochondrial dysfunction, inducing a classic ferroptosis mechanism. Marf overexpression or Drp1 knockdown protected against mitochondrial dysfunction and ferroptotic cell death in vitro. Furthermore, hypoxia-induced mitochondrial fission was verified to be driven by the Drp1/MTP18 interaction. Under hypoxia, MTP18 transcription was increased by the binding of activated HIF-1α to hypoxia response elements (HREs) in its promoter. Conjointly, MTP18 knockdown resulted in less apoptosis and decreased prawn mortality in gill tissue in vitro; suggesting that adaptation to hypoxia involves a vital function of MTP18. In conclusion, we uncovered a conserved role of mitochondrial fission in hypoxia-induced ferroptotic cell death. Therefore, we suggest that specific modulation of MTP18/DRP1-mediated mitochondrial dynamics might be a potential therapeutic strategy in hypoxic stress-induced tissue injury of invertebrates.

Antibodies are of central importance as reagents for the localization of proteins and other biomolecules in cells and tissues. To expand the repertoire of antibody-based reagents, we have constructed a series of plasmid vectors that permit expression of amino-terminal fusions to the hinge and Fc regions from goat, guinea pig, human, mouse, and rabbit IgGs, and chicken IgY. The resulting fusion proteins can be produced in transfected mammalian cells and detected with commercially available and species-specific secondary antibody reagents. We demonstrate the utility of this platform by constructing and testing Fc fusions with DARPin, single chain (sc)Fv, nanobody, toxin, and chemokine partners. The resulting fusion proteins were used to detect their targets in tissue sections or on the surface of transfected cells by immunofluorescent staining, or on the surface of immune cells by flow cytometry. By expanding the range of Fc sequences available for fusion protein production, this platform will expand the repertoire of primary antibody reagents for multiplexed immunostaining and FACS analyses.

Initiation of translation for the majority of eukaryotic mRNAs is mediated by a 5' cap structure to which the eukaryotic Initiation Factor 4E (eIF4E) binds. Inhibition of the activity of eIF4E by 4EBP-1 does not prevent the translation of a number of cellular capped mRNAs, indicative of the existence of previously unexplored mechanisms for the translation of these capped mRNAs without the requirement of eIF4E. eIF4G2, also known as Death Associated Protein 5 (DAP5), a homolog of eIFGI that lacks the eIF4E binding domain, utilizes eIF3d (a subunit of eIF3) to promote the translation of a subset of these mRNAs. Using fluorescence anisotropy-based equilibrium binding studies, we provide the first quantitative evidence of the recruitment of eIF3d as well as eIF3d and eIFG2 complexes to a subset of human mRNAs. Our quantitative studies demonstrate the critical role a fully methylated 5' mRNA cap structure plays in the recognition and recruitment of eIF3d, as well as the eIF3d and eIFG2 complex. By using luciferase reporter-based in vitro translation assays, we further show that cap-recognition ability correlates with the efficiency of translation of these mRNAs. Essentially, by preferably utilizing eIF3d and eIFG2, specific mRNA subsets are still able to translate in a cap-dependent manner even when eIF4E is sequestered. Our findings offer new insight into the use of eIF3d and eIF4G2 as an alternative for growth and survival under conditions of cellular stress. This novel mechanism of translation may offer new targets for therapeutic regulation of mRNA translation.