Microtubule-binding Protein Research Articles

Calumenin-1 Interacts with Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets.

SummaryThe ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.

Loss of CRMP2 O-GlcNAcylation leads to reduced novel object recognition performance in mice.

O-GlcNAcylation is an abundant post-translational modification in the nervous system, linked to both neurodevelopmental and neurodegenerative disease. However, the mechanistic links between these phenotypes and site-specific O-GlcNAcylation remain largely unexplored. Here, we show that Ser517 O-GlcNAcylation of the microtubule-binding protein Collapsin Response Mediator Protein-2 (CRMP2) increases with age. By generating and characterizing a Crmp2S517A knock-in mouse model, we demonstrate that loss of O-GlcNAcylation leads to a small decrease in body weight and mild memory impairment, suggesting that Ser517 O-GlcNAcylation has a small but detectable impact on mouse physiology and cognitive function.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

ObjectiveUnderstanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BackgroundTauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. New/Updated HypothesisWe hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. Major Challenges for the HypothesisOur approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. Linkage to Other Major TheoriesOur biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease–associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

A microtubule RELION-based pipeline for cryo-EM image processing

Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.

Nucleolar spindle associated protein 1 (NUSAP1) facilitates proliferation of hepatocellular carcinoma cells

Background: The most fundamental characteristic of cancer cells is abnormal proliferation, which is partly due to deregulation of cell cycle. Nucleolar spindle associated protein 1 (NUSAP1) is originally identified as microtubule and chromosome binding protein and takes an important role in chromosome segregation fidelity. Recent studies have been documented that NUSAP1 is associated with the progression of several cancers. However, its expressions and functions are unclear in hepatocellular carcinoma (HCC). Methods: qPCR and western blotting were used to examine NUSAP1 levels in fresh paired HCC tissues and corresponding adjacent normal liver tissues (ANT). MTT and colony formation assay were performed to evaluate whether NUSAP1 can influence the proliferation of HCC cells. Anchorage-independent growth assay was implemented to detect the effect of NUSAP1 on in vitro tumorigenicity capacity. Flow cytometry, qPCR and western blotting assay were used to clarify the detailed mechanisms that NUSAP1 accelerates the capability for cell proliferation and in vitro tumorigenicity. Results: Firstly, we analyzed the availably public dataset The Cancer Genome Atlas (TCGA), and discovered that NUSAP1 is dramatically upregulated in HCC tissues and closely correlated with poor prognosis of patients suffering HCC, which was confirmed in fresh HCC tissues and ANT. Subsequently, cell function assay illustrated that the upregulation of NUSAP1 facilitates proliferation and in vitro tumorigenicity capacity of HCC cells. Besides, molecular experiments suggested that NUSAP1 promotes cell cycle transition of HCC cells. Conclusions: In summary, the above results inferred that NUSAP1 takes an indispensable role in cell cycle progression. And it might be served as a novel prognostic factor in HCC.

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

Nucleolar spindle associated protein 1 (NUSAP1) facilitates proliferation of hepatocellular carcinoma cells.

BackgroundThe most fundamental characteristic of cancer cells is abnormal proliferation, which is partly due to deregulation of cell cycle. Nucleolar spindle associated protein 1 (NUSAP1) is originally identified as microtubule and chromosome binding protein and takes an important role in chromosome segregation fidelity. Recent studies have been documented that NUSAP1 is associated with the progression of several cancers. However, its expressions and functions are unclear in hepatocellular carcinoma (HCC).MethodsqPCR and western blotting were used to examine NUSAP1 levels in fresh paired HCC tissues and corresponding adjacent normal liver tissues (ANT). MTT and colony formation assay were performed to evaluate whether NUSAP1 can influence the proliferation of HCC cells. Anchorage-independent growth assay was implemented to detect the effect of NUSAP1 on in vitro tumorigenicity capacity. Flow cytometry, qPCR and western blotting assay were used to clarify the detailed mechanisms that NUSAP1 accelerates the capability for cell proliferation and in vitro tumorigenicity.ResultsFirstly, we analyzed the availably public dataset The Cancer Genome Atlas (TCGA), and discovered that NUSAP1 is dramatically upregulated in HCC tissues and closely correlated with poor prognosis of patients suffering HCC, which was confirmed in fresh HCC tissues and ANT. Subsequently, cell function assay illustrated that the upregulation of NUSAP1 facilitates proliferation and in vitro tumorigenicity capacity of HCC cells. Besides, molecular experiments suggested that NUSAP1 promotes cell cycle transition of HCC cells.ConclusionsIn summary, the above results inferred that NUSAP1 takes an indispensable role in cell cycle progression. And it might be served as a novel prognostic factor in HCC.

Sperm Flagellar 1 Binds Actin in Intestinal Epithelial Cells and Contributes to Formation of Filopodia and Lamellipodia

Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.

LIN9 confers paclitaxel resistance in triple negative breast cancer cells by upregulating CCSAP

LIN9 functions to regulate cell mitotic process. Dysregulation of LIN9 expression is associated with development of human cancers. In this study we assessed the association of LIN9 expression with paclitaxel resistance and clarified the underlying mechanisms for the first time. LIN9 expression in breast cancer tissues was retrieved from publicly available online databases and statistically analyzed. Human TNBC cell lines MDA-MB-231 and MDA-MB-468 and their corresponding paclitaxel-resistant sublines 231PTX and 468PTX were used to assess the expression of LIN9 by qRT-PCR and Western blot, cell growth by cell counting, cell viability by MTS assay, and cell apoptosis by flow cytometry. The data showed that high LIN9 expression in breast cancer patients receiving chemotherapy was related to poor overall survival (OS). LIN9 expression was upregulated in paclitaxel-resistant TNBC cells compared to their parental cells. Knockdown of LIN9 or treatment of paclitaxel-resistant TNBC cells with a bromo- and extra-terminal domain inhibitor (BETi) JQ1 which also decreased LIN9 expression enhanced the sensitivity of paclitaxel-resistant TNBC cells to paclitaxel. Mechanistically, decreased LIN9 in resistant cell lines reduced tumor cell viability, promoted multinucleated cells formation and induced tumor cell apoptosis, potentially by directly regulating microtubule-binding protein CCSAP. In conclusion, high LIN9 expression contributed to poor clinical outcomes and paclitaxel resistance in TNBC and BETi, targeting LIN9 expression, could be a reversible drug for PTX-resistant TNBC patients.

Abstract 918: Pre-B-cell Leukemia Homeobox Interacting Protein 1 is a Novel Regulator of Growth Signaling in the Heart

The cellular mechanisms that allow for cardiomyocyte growth in different directions may yield important information about the development of hypertrophic and dilated cardiomyopathies, diseases which include the widening and lengthening growth of cardiomyocytes, respectively. We have previously found that genetic manipulations of the mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathway cause cardiac hypertrophy and cardiomyocyte widening when the pathway is activated or dilated cardiomyopathy and cardiomyocyte lengthening when it is inhibited. The downstream effectors of the directional growth response controlled by these kinases have yet to be studied. Therefore, an unbiased phosphoproteomic screen was conducted on cardiomyocytes subjected to activation or inhibition of MEK1-ERK1/2 signaling. After validation of the screening hits with western and Phos-tag blots, Pre-B-cell leukemia homeobox interacting protein 1 (PBXIP1) emerged as an interesting target. Although it is known as a microtubule binding protein and regulator of proliferation in cancer cells, its function has never before been studied in the heart. Inhibition of MEK1-ERK1/2 increases phosphorylation of PBXIP1. However, activation of the pathway increases PBXIP1 protein expression, and as such we sought to determine the effects of PBXIP1 overexpression in the heart via adeno-associated virus 9 (AAV9) treatment. At four months of age, mice overexpressing PBXIP1 have significantly larger hearts than controls without a change in cardiac contractility. Intriguingly, histological analysis showed that cardiomyocytes highly infected with PBXIP1 AAV9 are significantly smaller in size compared to uninfected cells. Current studies are manipulating the PBXIP1 phosphorylation sites identified in the phosphoproteomic screen to determine the exact roles they may play in the cardiac growth response.

In vitro 0N4R tau fibrils contain a monomorphic β-sheet core enclosed by dynamically heterogeneous fuzzy coat segments

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Effects of geldanamycin on neurite outgrowth-related proteins and kinases in nerve growth factor-differentiated pheochromocytoma 12 cells

Heat shock protein 90 (HSP90) antagonists are currently being evaluated as potential anticancer drugs. However, adverse effects related to these drugs, such as fatigue and pain, suggest that they affect neurons. Therefore, to understand the influence of HSP90 inhibitors on neurons, we investigated the effects of geldanamycin, an HSP90 antagonist, on nerve growth factor (NGF)-differentiated pheochromocytoma 12 (PC12) cells, particularly, on the expression and phosphorylation of proteins and kinases in the NGF pathway. Geldanamycin significantly inhibited NGF-induced neurite outgrowth and phosphorylation of Akt and extracellular signal-related kinase 1/2 in PC12 cells. Furthermore, geldanamycin inhibited the phosphorylation of collapsin response mediator protein 2 and the expression of cyclin-dependent kinase 5 in the presence of NGF, but did not significantly affect the expression of glycogen synthase kinase 3β. These results suggest that geldanamycin influences microtubule-binding proteins and kinases relating to neurite outgrowth, thereby inducing neuronal impairment.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment.

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.

Bioinformatics analysis for heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure but little is known about the underlying mechanisms. Dahl salt‐sensitive (Dahl‐SS) rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF and controls were fed a normal‐salt diet (0.3% NaCl). Echocardiograms and electrocardiography (ECG) was performed to assess cardiac function. Diastolic dysfunction and preserved ejection fraction along with signs of heart failure was showed and QTc interval on ECG was prolonged (p<0.001) at 14 weeks of age. Hearts were harvested for gene array and label‐free quantitative proteomics. Bioinformatics analysis using DAVID website, GSEA software and BinGO plugin in Cytoscape environment. GO analysis showed microtubule binding and calcium dependent phospholipid binding protein were significant downregulated while oxidative stress response protein were upregulated (p<0.05, q<0.05). GSEA results indicated that gap junction, focal adhesion and actin cytoskeleton regulation gene sets were concentrated in upregulated transcripts (p<0.01, q<0.25) while cardiac muscle contraction and oxidative phosphorylation pathway were downregulated in proteomics (p<0.001, q<0.001). Western blot and PCR experiments showed significant changes in the expression of Dynamin‐1, Integrin‐α9 and MFF, which had important functions including cell stiffness regulation. Our study provides valuable information on the molecular functions and regulatory network of HFpEF, as well as new perspectives on the relationship between diastolic dysfunction and calcium stimulate, cytoskeleton stiffness and oxidative stress.Support or Funding InformationShenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Miro-dependent mitochondrial pool of CENP-F and its farnesylated C-terminal domain are dispensable for normal development in mice.

CENP-F is a large, microtubule-binding protein that regulates multiple cellular processes including chromosome segregation and mitochondrial trafficking at cytokinesis. This multiplicity of functions is mediated through the binding of various partners, like Bub1 at the kinetochore and Miro at mitochondria. Due to the multifunctionality of CENP-F, the cellular phenotypes observed upon its depletion are difficult to interpret and there is a need to genetically separate its different functions by preventing binding to selected partners. Here we engineer a CENP-F point-mutant that is deficient in Miro binding and thus is unable to localize to mitochondria, but retains other localizations. We introduce this mutation in cultured human cells using CRISPR/Cas9 system and show it causes a defect in mitochondrial spreading similar to that observed upon Miro depletion. We further create a mouse model carrying this CENP-F variant, as well as truncated CENP-F mutants lacking the farnesylated C-terminus of the protein. Importantly, one of these truncations leads to ~80% downregulation of CENP-F expression. We observe that, despite the phenotypes apparent in cultured cells, mutant mice develop normally. Taken together, these mice will serve as important models to study CENP-F biology at organismal level. In addition, because truncations of CENP-F in humans cause a lethal disease termed Strømme syndrome, they might also be relevant disease models.

Nanoparticle formulations in the diagnosis and therapy of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common age-related diseases that occurs because of the deposition of amyloid fibrils in a form of extracellular plaques containing β-amyloid peptide (Aβ) and tangles are found as intracellular deposit in the brain made up of twisted strands of aggregated microtubule binding protein. Scores of small molecule inhibitors have been designed for the treatment of AD. However some of these drugs cannot pass through the brain-blood-barrier (BBB). To overcome this problem, various nanoparticles (NPs) or nanomedicines (NMs) have been synthesized. These nanoparticles exploit the existing physiological mechanisms of passing through the BBB, including receptor- and adsorptive-mediated transcytosis that facilitate the transcellular transport of nanoparticle from the blood to the brain. During the last decades, varieties of nanoparticles that differ in the composition have been developed, and they have the potential application in the diagnostics and therapy of AD. The most common NP formulations that have major impact in the diagnosis and therapy of AD include polymeric NPs (PPs), gold NPs, gadolinium NPs, selenium NPs, protein-based NPs, polysaccharide-based NPs, etc. The goal of this review is to provide discussion of the application of different types of NP formulations in the diagnosis and therapy of AD.

Nucleolar spindle-associated protein 1 promotes tumorigenesis and predicts poor prognosis in human esophageal squamous cell carcinoma.

The microtubule binding protein, nucleolar spindle-associated protein 1 (NUSAP1), has a crucial function in mitosis and its expression is closely associated with carcinogenesis. Herein, we aimed to determine the function of NUSAP1 in the development of human esophageal squamous cell carcinoma (ESCC), and the association of NUSAP1 expression with ESCC. Immunohistochemical staining of ESCC tissue sections indicated that NUSAP1 was expressed to a higher degree in tumor tissues than in adjacent nontumor tissues. NUSAP1 levels were relevant closely to histological differentiation (P = 0.049). Overall survival was longer in patients with lower NUSAP1 levels ( P < 0.001). NUSAP1 expression ( P = 0.002), histological differentiation ( P < 0.001), tumor depth ( P = 0.045), lymph node metastases ( P < 0.001), and tumor-node-metastasis staging ( P = 0.008) were greatly associated with overall survival using univariate analysis. Multivariate analysis suggested that histological differentiation ( P = 0.014) and NUSAP1 expression ( P = 0.026) could be independent prognostic markers for ESCC. Additionally, the biological behavior of ESCC cells was investigated in vitro and in vivo. Suppression of NUSAP1 inhibited cellular proliferation and invasion, and induced cell cycle arrest and apoptosis in vitro. More importantly, knockdown of NUSAP1 led to inhibition of tumor formation in nude mice. These findings indicated that NUSAP1 is a potential prognostic biomarker in ESCC, and is an ESCC oncogene. Thus, NUSAP1 could represent a therapeutic target for ESCC.

Hypomorphic mutations of TRIP11 cause odontochondrodysplasia.

Odontochondrodysplasia (ODCD) is an unresolved genetic disorder of skeletal and dental development. Here, we show that ODCD is caused by hypomorphic TRIP11 mutations, and we identify ODCD as the nonlethal counterpart to achondrogenesis 1A (ACG1A), the known null phenotype in humans. TRIP11 encodes Golgi-associated microtubule-binding protein 210 (GMAP-210), an essential tether protein of the Golgi apparatus that physically interacts with intraflagellar transport 20 (IFT20), a component of the ciliary intraflagellar transport complex B. This association and extraskeletal disease manifestations in ODCD point to a cilium-dependent pathogenesis. However, our functional studies in patient-derived primary cells clearly support a Golgi-based disease mechanism. In spite of reduced abundance, residual GMAP variants maintain partial Golgi integrity, normal global protein secretion, and subcellular distribution of IFT20 in ODCD. These functions are lost when GMAP-210 is completely abrogated in ACG1A. However, a similar defect in chondrocyte maturation is observed in both disorders, which produces a cellular achondrogenesis phenotype of different severity, ensuing from aberrant glycan processing and impaired extracellular matrix proteoglycan secretion by the Golgi apparatus.

Investigating the Role of Charge-Altering Post-Translational Modifications on Tau Peptide Conformational Ensembles using Polarizable Molecular Dynamics Simulations

Aggregation of amyloidogenic proteins, such as the amyloid β-peptide, microtubule-binding protein tau, and α-synuclein, is linked to a number of human diseases. Amyloidogenic proteins unfold, aggregate, and self-assemble into highly stable, insoluble fibrils characterized by cross-β structure. Despite having unique primary sequences, these amyloidogenic proteins all undergo similar conformational changes, from disordered and only partially structured, to highly ordered in the fibril form. The forces driving such changes in structure are not completely characterized and a better understanding is needed to potentially combat a wide range of diseases that are currently incurable. In this study, we performed molecular dynamics simulations with the Drude polarizable protein force field to gain a better understanding of protein aggregation. We previously found that the explicit representation of electronic polarization was required to delineate mutation-specific unfolding pathways of the amyloid β-peptide. Building upon those findings, here we explore the impact of charge-altering post-translational modifications (PTMs) on the dynamics of tau, the protein that forms intracellular, neurofibrillary tangles in Alzheimer's disease. PTMs like acetylation of lysine and phosphorylation of serine and threonine are known to induce tau aggregation, but the underlying molecular mechanism remains unknown. By studying fragments of tau harboring these PTMs, we sought to gain novel insights into how changes in charge caused by PTMs shift the conformational ensembles of fragments of the tau protein. Polarizable simulations of these charge-altering PTMs shed light on how electrostatic interactions, including those between permanent and induced dipoles, affect side-chain properties, salt bridges, water and ion interactions, and changes in secondary structure.