Molecular Bases of Disease
Temperature instability of a mutation at a multidomain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome
ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited.Lack of ApoE inhibits ADan amyloidosis in a mouse model of familial Danish dementia
The Apolipoprotein E-ε4 allele (APOE-ε4) is the strongest genetic risk factor for late onset Alzheimer disease (AD). ApoE plays a critical role in amyloid-β (Aβ) accumulation in AD, and genetic deletion of the murine ApoE gene in mouse models results in a decrease or inhibition of Aβ deposition. The association between the presence of ApoE and amyloid in amyloidoses suggests a more general role for ApoE in the fibrillogenesis process. However, whether decreasing levels of ApoE would attenuate amyloid pathology in different amyloidoses has not been directly addressed.Ubiquitin-specific peptidase 14 maintains estrogen receptor α stability via its deubiquitination activity in endometrial cancer
USP14 deubiquitinates ERα to maintain its stability in ECEndometrial cancer (EC) is one of the common gynecological malignancies of which the incidence has been rising for decades. It is considered that continuously unopposed estrogen exposure is the main risk factor for EC initiation. Thus, exploring the modulation of estrogen/estrogen receptor α (ERα) signaling pathway in EC would be helpful to well understand the mechanism of EC development and find the potential target for EC therapy. Ubiquitin-specific peptidase 14 (USP14), a member of the proteasome-associated deubiquitinating enzyme family, plays a crucial role in a series of tumors.The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy
The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome.Loss of transcription factor EB dysregulates the G1/S transition and DNA replication in mammary epithelial cells
Triple-negative breast cancer (TNBC) poses significant challenges for treatment given the lack of targeted therapies and increased probability of relapse. It is pertinent to identify vulnerabilities in TNBC and develop newer treatments. Our prior research demonstrated that transcription factor EB (TFEB) is necessary for TNBC survival by regulating DNA repair, apoptosis signaling, and the cell cycle. However, specific mechanisms by which TFEB targets DNA repair and cell cycle pathways are unclear, and whether these effects dictate TNBC survival is yet to be determined.Suppression of amyloid-β fibril growth by drug-engineered polymorph transformation
Fibrillization of the protein amyloid β is assumed to trigger Alzheimer’s pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure.PLTP is a p53 target gene with roles in cancer growth suppression and ferroptosis
The tumor suppressor protein p53 suppresses cancer by regulating processes such as apoptosis, cell cycle arrest, senescence, and ferroptosis, which is an iron-mediated and lipid peroxide–induced cell death pathway. Whereas numerous p53 target genes have been identified, only a few appear to be critical for the suppression of tumor growth. Additionally, while ferroptosis is clearly implicated in tumor suppression by p53, few p53 target genes with roles in ferroptosis have been identified. We have previously studied germline missense p53 variants that are hypomorphic or display reduced activity.Stress response protein REDD1 promotes diabetes-induced retinal inflammation by sustaining canonical NF-κB signaling
Inflammation contributes to the progression of retinal pathology caused by diabetes. Here, we investigated a role for the stress response protein regulated in development and DNA damage response 1 (REDD1) in the development of retinal inflammation. Increased REDD1 expression was observed in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes, and REDD1 was essential for diabetes-induced pro-inflammatory cytokine expression. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented increased pro-inflammatory cytokine expression in response to hyperglycemic conditions.Sequential hydrolysis of FAD by ecto-5′ nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B2 into cells
Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5′ nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells.Intracellular pathogen Leishmania intervenes in iron loading into ferritin by cleaving chaperones in host macrophages as an iron acquisition strategy
Iron (Fe) sequestration is one of the most important strategies of the host to control the growth and survival of invading pathogens. Ferritin (Ft) plays a pivotal role in the sequestration mechanism of mammalian hosts by storing Fe. Recent evidence suggests that poly(rC)-binding proteins (PCBPs) act as chaperones for loading Fe into Ft. Incidentally, modulation of host PCBPs in respect to storing Fe in Ft during any infection remains unexplored. Among PCBPs, PCBP1 and PCBP2 are present in every cell type and involved in interacting with Ft for Fe loading.Intracerebroventricular dosing of N-sulfoglucosamine sulfohydrolase in mucopolysaccharidosis IIIA mice reduces markers of brain lysosomal dysfunction
Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed.A CACNA1C variant associated with cardiac arrhythmias provides mechanistic insights in the calmodulation of L-type Ca2+ channels
We recently reported the identification of a de novo single nucleotide variant in exon 9 of CACNA1C associated with prolonged repolarization interval. Recombinant expression of the glycine to arginine variant at position 419 produced a gain in the function of the L-type CaV1.2 channel with increased peak current density and activation gating but without significant decrease in the inactivation kinetics. We herein reveal that these properties are replicated by overexpressing calmodulin (CaM) with CaV1.2 WT and are reversed by exposure to the CaM antagonist W-13.Metal migration and subunit swapping in ALS-linked SOD1: Zn2+ transfer between mutant and wild-type occurs faster than the rate of heterodimerization
The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔGHet) between WT and ALS-mutant SOD1 in mismatched metalation states—where one subunit is metalated and the other is not—have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by “stealing” metal ions remains untested.Long noncoding RNA SNHG5 promotes podocyte injury via the microRNA-26a-5p/TRPC6 pathway in diabetic nephropathy
Podocyte injury is a characteristic pathological hallmark of diabetic nephropathy (DN). However, the exact mechanism of podocyte injury in DN is incompletely understood. This study was conducted using db/db mice and immortalized mouse podocytes. High-throughput sequencing was used to identify the differentially expressed long noncoding RNAs in kidney of db/db mice. The lentiviral shRNA directed against long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) or microRNA-26a-5p (miR-26a-5p) agomir was used to treat db/db mice to regulate the SNHG5/miR-26a-5p pathway.Channel HCN4 mutation R666Q associated with sporadic arrhythmia decreases channel electrophysiological function and increases protein degradation
Mutations in the hyperpolarization-activated nucleotide-gated channel 4 (HCN4) are known to be associated with arrhythmias in which QT prolongation (delayed ventricular repolarization) is rare. Here, we identified a HCN4 mutation, HCN4-R666Q, in two sporadic arrhythmia patients with sinus bradycardia, QT prolongation, and short bursts of ventricular tachycardia. To determine the functional effect of the mutation, we conducted clinical, genetic, and functional analyses using whole-cell voltage-clamp, qPCR, Western blot, confocal microscopy, and co-immunoprecipitation.Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death
Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle–like “LAM” cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear.Aging-associated REGγ proteasome decline predisposes to tauopathy
The REGγ-20S proteasome is an ubiquitin- and ATP-independent degradation system, targeting selective substrates, possibly helping to regulate aging. The studies we report here demonstrate that aging-associated REGγ decline predisposes to decreasing tau turnover, as in a tauopathy. The REGγ proteasome promotes degradation of human and mouse tau, notably phosphorylated tau and toxic tau oligomers that shuttle between the cytoplasm and nuclei. REGγ-mediated proteasomal degradation of tau was validated in 3- to 12-month-old REGγ KO mice, REGγ KO;PS19 mice, and PS19 mice with forebrain conditional neuron-specific overexpression of REGγ (REGγ OE) and behavioral abnormalities.BTK kinase activity is dispensable for the survival of diffuse large B-cell lymphoma
Inhibitors targeting Bruton's tyrosine kinase (BTK) have revolutionized the treatment for various B-cell malignancies but are limited by acquired resistance after prolonged treatment as a result of mutations in BTK. Here, by a combination of structural modeling, in vitro assays, and deep phospho-tyrosine proteomics, we demonstrated that four clinically observed BTK mutations—C481F, C481Y, C481R, and L528W—inactivated BTK kinase activity both in vitro and in diffused large B-cell lymphoma (DLBCL) cells.Wnt signaling modulator DKK4 inhibits colorectal cancer metastasis through an AKT/Wnt/β-catenin negative feedback pathway
Aberrant activation of the Wnt/β-catenin signaling pathway is implicated in most malignant cancers, especially in the initiation and progression of colorectal cancer (CRC). DKK4 is a classical inhibitory molecule of the Wnt/β-catenin pathway, but its role in CRC is ambiguous, and the molecular mechanism remains unclear. Here, we determined DKK4 expression was significantly upregulated in 23 CRC cell lines and 229 CRC tissues when analyzed by quantitative PCR and immunohistochemistry, respectively.Intermittent hypoxia enhances the expression of hypoxia inducible factor HIF1A through histone demethylation
The cellular response to hypoxia is regulated through enzymatic oxygen sensors, including the prolyl hydroxylases, which control degradation of the well-known hypoxia inducible factors (HIFs). Other enzymatic oxygen sensors have been recently identified, including members of the KDM histone demethylase family. Little is known about how different oxygen-sensing pathways interact and if this varies depending on the form of hypoxia, such as chronic or intermittent. In this study, we investigated how two proposed cellular oxygen-sensing systems, HIF-1 and KDM4A, KDM4B, and KDM4C, respond in cells exposed to rapid forms of intermittent hypoxia (minutes) and compared to chronic hypoxia (hours).Distinct neurotoxic TDP-43 fibril polymorphs are generated by heterotypic interactions with α-Synuclein
Amyloid aggregates of specific proteins constitute important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show comorbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by colocalizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids.The atypical Rab GTPase associated with Parkinson’s disease, Rab29, is localized to membranes
Several genetic studies have shown that the small GTPase Rab29 is involved in the pathogenesis of Parkinson’s Disease (PD). It has also been shown that overexpression of Rab29 increases the activity of leucine-rich repeat kinase 2, a protein kinase often mutated in familial PD, although the mechanism underlying this activation remains unclear. Here, we employed biochemical analyses to characterize the localization of Rab29 and found that, unlike general Rab proteins, Rab29 is predominantly fractionated into the membrane fraction by ultracentrifugation.Natural and engineered inflammasome adapter proteins reveal optimum linker length for self-assembly
The inflammasome is a multiprotein complex that triggers the activation of proinflammatory cytokines. The adapter ASC and its isoform ASCb mediate inflammasome assembly via self-association and oligomerization with other inflammasome proteins by homotypic interactions of their two identical Death Domains, PYD and CARD, connected by a linker of different length: 23 (ASC) and 4 (ASCb) amino acids long. However, ASC is a more potent inflammasome activator compared to ASCb. Thus, adapter isoforms might be involved in the regulation of the inflammatory response.Glucocorticoid receptor dysregulation underlies 5-HT2AR-dependent synaptic and behavioral deficits in a mouse neurodevelopmental disorder model
Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring.MicroRNA-16 inhibits the TLR4/NF-κB pathway and maintains tight junction integrity in irritable bowel syndrome with diarrhea
Irritable bowel syndrome with diarrhea (IBS-D) is a chronic and relapsing inflammatory disorder in which pathogenesis has been shown to be in part the result of miRNA-mediated signaling. Here, we investigated the alleviatory role of miR-16 in IBS-D. First, we established an IBS-D mouse model using colonic instillation of acetic acid and developed an IBS-D cell model using lipopolysaccharide exposure. The experimental data demonstrated that miR-16 was underexpressed in the serum of IBS-D patients, as well as in the colorectal tissues of IBS-D mouse models and lipopolysaccharide-exposed intestinal epithelial cells.
