The Journal of Biological Chemistry最新文献

Hyperosmolarity of the ocular surface triggers inflammation and pathological damage in dry eye disease (DED). In addition to a reduction in quality of life, DED causes vision loss and when severe, blindness. Mitochondrial dysfunction occurs as a consequence of hyperosmolar stress. We have previously reported on a role for the insulin-like growth factor binding protein-3 (IGFBP-3) in the regulation of mitochondrial ultrastructure and metabolism in mucosal surface epithelial cells; however, this appears to be context-specific. Due to the finding that IGFBP-3 expression is decreased in response to hyperosmolar stress in vitro and in an animal model of DED, we next sought to determine whether the hyperosmolar stress-mediated decrease in IGFBP-3 alters mitophagy, a key mitochondrial quality control mechanism. Here we show that hyperosmolar stress induces mitophagy through differential regulation of BNIP3L/NIX and PINK1-mediated pathways. In corneal epithelial cells, this was independent of p62. The addition of exogenous IGFBP-3 abrogated the increase in mitophagy. This occurred through regulation of mTOR, highlighting the existence of a new IGFBP-3-mTOR signaling pathway. Together, these findings support a novel role for IGFBP-3 in mediating mitochondrial quality control in DED and have broad implications for epithelial tissues subject to hyperosmolar stress and other mitochondrial diseases.

The extracellular signal-regulated kinase (ERK) controls multiple critical processes in the cell and is deregulated in human cancers, congenital abnormalities, immune diseases, and neurodevelopmental syndromes. Catalytic activity of ERK requires dual phosphorylation by an upstream kinase, in a mechanism that can be described by two sequential Michaelis-Menten steps. The estimation of individual reaction rate constants from kinetic data in the full mechanism has proved challenging. Here, we present an analytically tractable approach to parameter estimation that is based on the phase plane representation of ERK activation and yields two combinations of six reaction rate constants in the detailed mechanism. These combinations correspond to the ratio of the specificities of two consecutive phosphorylations and the probability that monophosphorylated substrate does not dissociate from the enzyme before the second phosphorylation. The presented approach offers a language for comparing the effects of mutations that disrupt ERK activation and function in vivo. As an illustration, we use phase plane representation to analyze dual phosphorylation under heterozygous conditions, when two enzyme variants compete for the same substrate.

Neurodegenerative tauopathies are caused by the transition of tau protein from a monomer to a toxic aggregate. They include Alzheimer disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick disease (PiD). We have previously proposed that tau monomer exists in two conformational ensembles: an inert form (M_i), which does not self-assemble, and seed-competent form (M_s), which self-assembles and templates ordered assembly growth. We proposed that cis/trans isomerization of tau at P301, the site of dominant disease-associated S/L missense mutations, might underlie the transition of wild-type tau to a seed-competent state. Consequently, we created monoclonal antibodies using non-natural antigens consisting of fluorinated proline (P∗) at the analogous P270 in repeat 1 (R1), biased toward the trans-configuration at either the R1/R2 (TENLKHQP∗GGGKVQIINKK) or the R1/R3 (TENLKHQP∗GGGKVQIVYK) interfaces. Two antibodies, MD2.2 and MD3.1, efficiently immunoprecipitated soluble seeds from AD and PSP but not CBD or PiD brain samples. The antibodies efficiently stained brain samples of AD, PSP, and PiD, but not CBD. They did not immunoprecipitate or immunostain tau from the control brain. Creation of potent anti-seed antibodies based on the trans-proline epitope implicates local unfolding around P301 in pathogenesis. MD2.2 and MD3.1 may also be useful for therapy and diagnosis.

The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome. Mechanistically, cytoplasmic NELFB physically and functionally interacts with prosurvival signaling kinases, most notably phosphatidylinositol-3-kinase/AKT. Ectopic expression of membrane-tethered phosphatidylinositol-3-kinase/AKT partially bypasses the role of NELFB in cell proliferation, but not Pol II occupancy. Together, these data expand the current understanding of the physiological impact of Pol II pausing and underscore the multiplicity of the biological functions of individual NELF subunits.

The kinesin-14 motor proteins play important roles in tumor development and drug resistance and have been reported as potential biomarkers or therapeutic targets for tumor treatment. However, kinesin family member C2 (KIFC2), one of the kinesin-14 motor family members, remains largely unknown in prostate cancer (PCa) progression. Here, we used the GEO and The Cancer Genome Atlas datasets, Western blotting, and immunohistochemistry analyses to detect KIFC2 expression in PCa tissues. Additionally, a series of in vivo and in vitro experiments were utilized to demonstrate the roles of KIFC2 in PCa cells. We found that KIFC2 was highly expressed and positively correlated with the clinicopathological characteristics in PCa. Functional experiments indicated that KIFC2 could promote PCa progression. Furthermore, we performed an analysis of the KEGG and GSEA databases, subcellular fractionation, and immunofluorescence to investigate the potential mechanisms of KIFC2 in PCa. We confirmed that KIFC2 could regulate the NF-κB pathway via mediating NF-κB p65 protein expression and nuclear translocation thereby promoting PCa progression and chemotherapeutic resistance. Together, our results suggest that KIFC2 is overexpressed in PCa. By regulating the NF-κB pathway, KIFC2 may play a crucial role in PCa.

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3C^pro protease, and 3D^pol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3C^pro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABC^pro becomes 20-fold more active than 3C^pro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

9-cis-retinoic acid (9cRA) binds retinoic acid receptors (RAR) and retinoid X receptors (RXR) with nanomolar affinities, in contrast to all-trans-retinoic acid (atRA), which binds only RAR with nanomolar affinities. RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRA occurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas β-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7 mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the β-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRA in vivo, and its self-sufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca²⁺ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

The regulation of translation provides a rapid and direct mechanism to modulate the cellular proteome. In eukaryotes, an established model for the recruitment of ribosomes to mRNA depends upon a set of conserved translation initiation factors. Nevertheless, how cells orchestrate and define the selection of individual mRNAs for translation, as opposed to other potential cytosolic fates, is poorly understood. We have previously found significant variation in the interaction between individual mRNAs and an array of translation initiation factors. Indeed, mRNAs can be separated into different classes based upon these interactions to provide a framework for understanding different modes of translation initiation. Here, we extend this approach to include new mRNA interaction profiles for additional proteins involved in shaping the cytoplasmic fate of mRNAs. This work defines a set of seven mRNA clusters, based on their interaction profiles with 12 factors involved in translation and/or RNA binding. The mRNA clusters share both physical and functional characteristics to provide a rationale for the interaction profiles. Moreover, a comparison with mRNA interaction profiles from a host of RNA binding proteins suggests that there are defined patterns in the interactions of functionally related mRNAs. Therefore, this work defines global cytoplasmic mRNA binding modules that likely coordinate the synthesis of functionally related proteins.