Journal of Biological Chemistry最新文献

Staufen2 (STAU2) is an RNA binding protein that controls mRNA trafficking and expression. Previously, we showed that its paralog Staufen1 (STAU1) was overabundant in cellular and mouse models of neurodegenerative diseases and amyotrophic lateral sclerosis (ALS) patient spinal cord. Here we investigated features of STAU2 that might parallel STAU1. STAU2 protein, but not mRNA, was overabundant in spinocerebellar ataxia type 2 (SCA2), ALS/frontotemporal dementia (FTD) patient fibroblasts, ALS patient spinal cord tissues, and in central nervous system (CNS) tissues from SCA2 and ALS animal models. Exogenous expression of STAU2 in HEK293 cells activated mechanistic target of rapamycin (mTOR) and stress granule formation. Targeting STAU2 by RNAi normalized mTOR in SCA2 and C9ORF72 cellular models. The microRNA miR-217, previously identified as downregulated in SCA2 mice, targets the STAU2 3'-UTR. We now demonstrate that exogenous expression of miR-217 significantly reduced STAU2 and mTOR levels in cellular models of neurodegenerative disease. These results suggest a functional link between STAU2 and mTOR signaling and identify a major role for miR-217 that could be exploited in therapeutic development.

Nitric oxide (NO) signaling often relies on it activating cGMP production by the heterodimeric enzyme soluble guanylyl cyclase (sGC). To mature to function an sGCβ subunit must first incorporate heme and then form a heterodimer with a partner α subunit. Our previous studies in cells showed that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) supplies heme to the apo-sGCβ subunit, which is complexed with the cell chaperone Hsp90. Through its ATP hydrolysis, Hsp90 then promotes heme insertion into apo-sGCβ and consequent formation of a functional heterodimer. NO at physiologic levels somehow stimulates cell heme allocation into apo-sGCβ by this process. To gain insight we utilized purified apo-sGCβ and GAPDH reporter proteins whose heme contents can be followed by fluorescence and determined the impact of Hsp90 and NO on heme transfer between them. Results show that heme transfer out of GAPDH and into apo-sGCβ is tightly coupled in all circumstances and is limited by the ability of the apo-sGCβ to incorporate the heme, which in turn relies on a ferric to ferrous heme transition taking place inside the sGCβ. Hsp90 can influence the heme transfer kinetics in a negative or positive manner through its conformational effects on apo-sGCβ, while NO speeds heme transfer by binding to the heme iron and thus speeding heme dissociation from GAPDH. Our findings provide new mechanistic understanding of sGC maturation and how Hsp90 and NO combine to dynamically regulate heme incorporation for sGC heterodimer formation and consequent cGMP production in biological settings.

Ricin is a Category B agent for bioterrorism and Shiga toxins are the primary virulence factors of Shiga toxin (Stx) producing E. coli (STEC). Ricin and Stxs bind the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop (SRL) on the eukaryotic ribosome and inhibit translation. Both toxins are prime targets for therapeutic intervention because no effective therapy exists for ricin intoxication or STEC infection. Binding of ricin toxin A subunit (RTA) to the ribosomal P-stalk stimulates depurination of the SRL by an unknown mechanism. We previously identified compounds that bind the P-stalk pocket of RTA and inhibit catalytic activity. Here we characterize a second-generation lead compound, which binds the P-stalk pocket of RTA with over 30-fold improved affinity relative to the original compound and inhibits the cytotoxicity of ricin holotoxin in Vero cells with no apparent cellular toxicity by itself. This compound also shows protection against Stx2A1. X-ray crystal structure of RTA-inhibitor complexes suggests that the orientation of the carboxylic acid influences the inhibitor contacts at the P-stalk site of RTA and contributes to inhibitor potency. The structural changes triggered at the P-stalk site of RTA were validated by solution NMR based chemical shift perturbation analysis. A key finding by NMR is that binding induced conformational changes extend beyond the P-stalk site to residues in the active site cleft of RTA. Collectively, these results provide valuable new insight into the conformational flexibility in the C-terminal domain of RTA and its potential role in mediating the remarkable catalytic activity of ricin.

The trafficking and aggregation of neurodegenerative proteins often involves the interaction between intrinsically disordered domains, stabilized by the inclusion of physiologic metal ions such as copper or zinc. Characterizing the metal ion coordination environment is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order or global structure. The cellular prion protein (PrP^C) binds both monomers and aggregates of Alzheimer's amyloid-beta (Aβ), promoting Aβ internalization and aberrant signaling, respectively. Both proteins bind Cu²⁺ with high affinity, opening the potential for copper to form an intermolecular bridge. We describe here a novel approach utilizing multiple EPR experiments to investigate the simultaneous Cu²⁺ coordination of PrP^C and Aβ in a 1:1:1 mixture. Uniformly ¹⁵N-labeled PrP^C is used in conjunction with natural abundance ¹⁴N Aβ, the combination of which leads to distinct energy manifolds for paramagnetic Cu²⁺ and resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize ¹⁴N (I = 1) and ¹⁵N (I = ½) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple ¹⁵N imidazole coordination. This unique approach provides compelling evidence of a copper-stabilized ternary complex, with equatorial Cu²⁺ coordination formed by one histidine imidazole from Aβ and three from PrP. Moreover, the methodologies developed here provide a framework for assessing the copper environment in other interacting neurodegenerative proteins.

Microglial activation is the initial pathological event that occurs in demyelination, a prevalent feature in various neurological diseases. G protein-coupled estrogen receptor (GPER1), which is highly expressed in microglia, has been reported to reduce myelin damage. However, the precise molecular mechanisms involved remain unclear. In this study, the cuprizone (CPZ) -induced demyelination model was used to investigate the relationship between GPER1 and myelin sheath injury and its mechanism. The results demonstrated that GPER1 deficiency exacerbated cognitive impairment in mice. Along with more severe myelin damage as well as fewer oligodendrocytes. Moreover, GPER1 deficiency not only directly reduced the number of microglia in CPZ mice, but also caused iron ions overload in microglia of myelin debris induced in vitro. Transcriptomic, molecular biological, and morphological analyses revealed that microglial ferroptosis caused by GPER1 deficiency contributes to the reduction of microglia number. In summary, these findings revealed that GPER1 can regulate demyelination through ferroptosis of microglia.

Lung cancer presents the highest mortality rate in the world when compared to other cancer types and often presents chemotherapy resistance to cisplatin. The A549 non-small cell lung cancer (NSCLC) line is widely used as a model for lung adenocarcinoma studies since it presents a high proliferative rate and a nonsense mutation in the STK11 gene. The LKB1 protein, encoded by the STK11 gene, is one of the major regulators of cellular metabolism through AMPK activation under nutrient deprivation. Mutation in the STK11 gene in A549 cells potentiates cancer hallmarks, such as deregulation of cellular metabolism, aside from the Warburg effect, mTOR activation, autophagy inhibition, and NRF2 and redox activation. In this study, we investigated the integration of these pathways associated with the metabolism regulation by LKB1/AMPK to improve cisplatin response in the A549 cell line. We first used the CRISPR/Cas9 system to generate cell lines with a CRISPR-edited LKB1 isoform (called Super LKB1), achieved through the introduction of a +1 adenine insertion in the first exon of the STK11 gene after NHEJ mediated repair. This insertion led to the expression of a higher molecular weight protein containing an alternative exon described in the Peutz-Jeghers Syndrome (PJS). Through metabolic regulation by Super LKB1 expression and AMPK activation, we found an increase in autophagy flux (LC3 GFP/RFP p<0.05), as well as a reduction in the phosphorylation of mTORC1 downstream targets (S6K2 phospho-Serine 423; p<0.05; and S6 ribosomal protein phospho-Serine 240/244; p<0.03). The NRF2 protein exhibited increased levels and more nuclear localization in A549 WT (wild-type) cells compared to the edited cells (p<0.01). We also observed lower levels of H₂O₂ in the WT A549 cells, as a possible result of NRF2 activation, and a higher requirement of cisplatin to achieve the IC₅₀ (WT: 10 μM; c2SL+: 5.5 μM; c3SL+: 6 μM). The data presented here suggests that the regulation of molecular pathways by the novel Super LKB1 in A549 cells related to metabolism, mTORC1, and autophagy promotes a better response of lung cancer cells to cisplatin. This NHEJ-CRISPR-based approach may be potentially used for lung cancer gene therapy.

Metabolic alterations in the somatosensory cortex play a crucial role in neuropathic pain development, as evidenced by magnetic resonance spectroscopy and mass spectrometry analyses of brain homogenates. However, investigating metabolic changes in specific neuronal subtypes during neuropathic pain development remains challenging. Here, utilizing a recently developed technique called single-cell mass spectrometry (SCMS), we investigated metabolomic alterations within excitatory glutamatergic neurons located in the primary somatosensory cortex (S1) during various stages of neuropathic pain. Specifically, we induced neuropathic pain in mice using a spared nerve injury (SNI) model and observed activation of glutamatergic neurons in layer II/III of S1 through c-Fos staining and electrophysiology. We profiled metabolic changes and performed pathway enrichment analysis in these neurons by SCMS during both acute and sub-chronic phases of SNI. Further analyses revealed metabolites whose alterations significantly correlated with changes in pain thresholds, as well as distinct temporal patterns of metabolite expression during pain progression. From these analyses, we identified several key metabolites (homogentisic acid, phosphatidylcholine, phosphorylcholine and rhein) and validated their causal roles in pain modulation via pharmacological interventions. Thus, our study provides a valuable resource for elucidating the neurometabolic regulatory mechanisms underlying neuropathic pain from a single-cell perspective.

Evidence suggests that ARV1 regulates sterol movement within the cell. Saccharomyces cerevisiae cells lacking ScArv1 have defects in sterol trafficking, distribution, and biosynthesis. HepG2 cells treated with hARV1 anti-sense oligonucleotides accumulate cholesterol in the endoplasmic reticulum. Mice lacking Arv1 have a lean phenotype when fed a high fat diet and show no signs of liver triglyceride or cholesterol accumulation, suggesting a role for Arv1 in lipid transport. Here, we explored the direct lipid binding activity of recombinant human ARV1 using in vitro lipid binding assays. ARV1 lipid binding activity was observed within the first N-terminal 98 amino acids containing the conserved ARV1 homology domain (AHD). The zinc-binding domain and conserved cysteine clusters within the AHD were necessary for lipid binding. Both full-length ARV1 and the AHD bound cholesterol, several phospholipids, and phosphoinositides with high affinity. The AHD showed the highest binding affinity for monophosphorylated phosphoinositides. Several conserved amino acids within the AHD were necessary for phospholipid binding. Biochemical studies suggested that ARV1 exists as a dimer in cells, with oligomerization being critical for ARV1 function, as amino acid mutations predicted to have a negative effect on dimerization cause weakened or complete loss of lipid binding. Our results show for the first time that human ARV1 can directly bind cholesterol and phospholipids. How this activity may function to regulate lipid binding and maintain proper lipid trafficking and/or transport in cells requires further studies.

Secretory protein expression in mammalian cells is widely used in various fields, including biomedical research and biopharmaceutical production. However, achieving high-level expression of certain secretory proteins / peptides can be challenging. The naturally occurring N1 fragment of the prion protein is one of these difficult-to-produce secretory proteins, which hinders our understanding of its biological functions and limits its potential as a therapeutic molecule. To improve N1 production, we screened several well-folded protein domains and found that fusing N1 with a camelid nanobody (Nb) improved its translocation into the endoplasmic reticulum and significantly enhanced its secretion. Nb-fusion does not alter the translocation mechanism, which remains dependent on the Sec61/Sec62/Sec63 complex. This approach also resulted in a significant increase in N1 production in the mouse brain using recombinant adeno-associated virus. Furthermore, fusing Nb to another unstructured protein, Shadoo (without GPI anchor), or a peptide hormone, somatostatin, also greatly increased their production, demonstrating the applicability of this approach to other proteins and peptides. The enhancement of N1 production is comparable or better than Fc fusion, and the effect is observed with all tested camelid Nb, but not with a shark Nb and to a lesser extent, with a human immunoglobulin heavy chain variable region. Importantly, the Nb in the fusion protein retained its antigen-binding capability, paving the way for the development of a dual-functional protein. Collectively, we present a novel strategy for enhancing the production of secretory proteins, which holds great promise in creating functional biological molecules for a wide range of applications.

Vitiligo, an autoimmune disease caused by environmental and genetic factors, is characterized by the specific loss of epidermal melanocytes (MCs). IFN-γ, predominantly derived from MC-targeting CD8⁺ T cells, plays a key role in vitiligo pathogenesis. Previously, we found that glycoprotein nonmetastatic melanoma protein B (GPNMB) is specifically lost in the basal epidermal layer of vitiligo lesions and downregulated by IFN-γ in normal human epidermal keratinocytes (KCs) (NHEKs). This study aimed to determine the role of KC GPNMB in normal and vitiligo epidermis and demonstrated that GPNMB plays a protective role against H₂O₂-induced oxidative stress due to its extracellular domain. In contrast, the NRF2/KEEP1 system was not involved in the anti-oxidative response in NHEKs but was active in MCs. GPNMB knockdown reduced the phosphorylation levels of AKT^T308 and AKT^S473 after H₂O₂ treatment, accompanied by reduced Dickkopf-1 (DKK1) mRNA and protein production and decreased FOXM1 mRNA expression. These results suggested that GPNMB protects KCs from H₂O₂-induced cell death through enhanced PI3K/AKT signaling, and WNT/β-catenin/FOXM1 and DKK1/CKAP4/AKT pathways. Furthermore, a significant increase in thioredoxin-interacting protein (TXNIP) following GPNMB knockdown was observed, indicating the enhanced phosphorylation of JNK and p38 and suppression of WNT/β-catenin signaling. These results suggest that the decreased expression of epidermal GPNMB in vitiligo lesions triggers increased sensitivity to H₂O₂-induced oxidative stress and decreased WNT/β-catenin signaling, consistent with the pathological features of the vitiligo epidermis. These findings may enhance our understanding of vitiligo pathogenesis, provide insights into the reduced risk of epidermal cancers, and highlight novel targets for treatment.