Subcellular Protein Research Articles

Novel POLE mutations identified in patients with IMAGE-I syndrome cause aberrant subcellular localisation and protein degradation in the nucleus

BackgroundDNA replisome is a molecular complex that plays indispensable roles in normal DNA replication. IMAGE-I syndrome is a DNA replisome-associated genetic disease caused by biallelic mutations in the gene encoding...

Subcellular Protein Expression of Annexin A1 (ANXA1) in Different Types of Cancer

Annexin A1 (ANXA1) is a Ca2+‐regulated phospholipid‐binding protein involved in several physiological functions. Further research has shown that this protein might have various roles in cancer development and operate at different levels. This is partially related to the placement of ANXA1 in distinct cellular compartments. ANXA1 is maybe nuclear, cytoplasmic, and membrane‐associated. The goal of this study has been to analyze ANXA1 subcellular localization and expression in different types of cancer.MethodsWe examined 1295 data sets available in The Human Protein Atlas (HPA) (HPA). The research examined the ANXA1 expression in tissue from 1130 cancer and 165 normal samples. A visual categorization method based on staining intensity (I), the proportion of stained cells (F) (F). ANXA1 expression levels as a function of subcellular localization.Q Score (product of E*I) was calculated by the fraction of stained cells (F) and the intensity (I) (I)ResultsIn cancer patients, the predominant subcellular localization was cytoplasmic membranous (53.3 %), whereas, in healthy individuals, the principal was cytoplasmic membranous and nuclear (57.0 %). In terms of staining intensity (Q Score), the highest values were found at the cytoplasmic membranous and nuclear levels (Q Score=5.69). In contrast, the highest Q Score values were found in standard samples at the nuclear level (Q Score=6.0).ConclusionThe subcellular localization of ANXA1 expression may be a helpful diagnostic marker in various cancer types.

Prediction of Proteins Subcellular Location in Microscopic Images Using Fine-Tuned Deep Learning Architectures

Understanding protein behavior depends heavily on where a protein is located within the cell. The successful prediction of protein locations can assess the early diagnosis of a disease and the effectiveness of various drugs. As a result, it is critical to create a protein localization prediction system that is both dependable and accurate. Numerous methods and resources have been employed to obtain insight into protein function over the years. Confocal Microscopy, employed by Human Protein Atlas, is one of them. In this research, the focus has been made on the localization of proteins in 19 subcellular compartments in human cells. The objective is to determine the organelle of the cell that contains the protein. In this research, a deep learning method for categorizing subcellular protein patterns in human cells is presented. The proposed method is primarily based on transfer learning technique using two pre-trained models, VGG-16 and DenseNet121. Due to the highly unbalanced nature of the dataset utilized in this study, class weight optimization is used before training the model. The model's performance is measured using the F1 score, with a final score of 0.75 for VGG-16 and 0.68 for DenseNet121.

Decoding the Therapeutic Implications of the ERα Stability and Subcellular Distribution in Breast Cancer.

Approximately 70% of all breast cancer cases are estrogen receptor-alpha positive (ERα+) and any ERα signaling pathways deregulation is critical for the progression of malignant mammary neoplasia. ERα acts as a transcription factor that promotes the expression of estrogen target genes associated with pro-tumor activity in breast cancer cells. Furthermore, ERα is also part of extranuclear signaling pathways related to endocrine resistance. The regulation of ERα subcellular distribution and protein stability is critical to regulate its functions and, consequently, influence the response to endocrine therapies and progression of this pathology. This minireview highlights studies that have deciphered the molecular mechanisms implicated in controlling ERα stability and nucleo-cytoplasmic transport. These mechanisms offer information about novel biomarkers, therapeutic targets, and promising strategies for breast cancer treatment.

Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images.

Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses.

An ex vivo approach to understanding sex-specific differences in placental androgen signalling in the presence and absence of inflammation.

The mechanisms that contribute to continued male intrauterine growth in response to an adverse maternal environment, such as those brought on by maternal asthma, remain largely undefined but may, in part, be mediated by androgen-mediated signalling. We previously reported the expression of multiple AR protein isoforms in the human placenta and proposed the novel AR-45 isoform to be integral in mediating male-specific androgen-dependent signalling in the presence of maternal asthma. In the current study we have used an ex vivo approach to further understand sex-specific differences in placental androgen signalling in the presence and absence of inflammation using human term villous placental explants. Explants were cultured in the presence and absence of 0.1nM dihydrotestosterone (DHT), 1μg/ml lipopolysaccharide (LPS), or DHT+LPS for 24hr. Tissue was used for gene expression and subcellular AR protein isoform expression. Cytoplasmic and nuclear AR protein isoforms expression did not vary between culture conditions in either sex. AR-45 activity was upregulated in male placentae cultured in DHT, LPS and DHT+LPS only. There were no changes in the expression of androgen-mediated downstream targets in males in response to culture conditions, but females had significantly reduced IGF1R expression in response to LPS. Increased AR-45 activity in the presence of inflammation may drive continued male feto-placental growth via maintained expression of downstream growth targets. Our findings build on previous work suggesting an important role for AR-45 in regulating male-specific adaptations to placental inflammation and underscores the need to further characterise the function of this AR isoform.

Optogenetic Application to Investigating Cell Behavior and Neurological Disease.

Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways (e.g., agonist/antagonist addition, overexpression, silencing, knockouts) are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field.

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

LncRNA NBAT-1 inhibits the progression of hepatocellular carcinoma by interacting with CYLD

Purpose: To illustrate the biological influences of long non-coding RNA (lncRNA) NBAT-1 (neuroblastoma associated transcript 1) on HCC progression and the molecular mechanism of action.
 Methods: NBAT-1 levels in HCC tissues and cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between NBAT-1 and prognosis in HCC was analyzed. After knockdown of NBAT-1 in HepG2 and Hep3B cells, proliferative and migratory changes were assessed by cell counting kit-8 (CCK-8) and Transwell assay, respectively. The interaction between NBAT-1 and CYLD was confirmed by subcellular fraction determination and RNA binding protein immunoprecipitation (RIP). Rescue experiments were conducted to verify the involvement of CYLD in HCC cell functions regulated by NBAT-1.
 Results: NBAT-1 was downregulated in HCC tissues. Its level was much lower in metastatic or advanced stage HCC patients (p < 0.05), showing a certain prognostic potential. Knockdown of NBAT-1 stimulated proliferative and migratory potentials in HepG2 and Hep3B cells. NBAT-1 was mainly distributed in the cell cytoplasm. The mRNA and protein levels of CYLD were downregulated in HCC cells by NBAT-1 knockdown, displaying a positive interaction. CYLD was involved in the regulatory effect of NBAT-1 on HCC progression.
 Conclusion: Through a positive interaction with CYLD, NBAT-1 inhibits the malignant progression of HCC. These findings provide a potential approach to the development of targeted therapies for HCC.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

NanoBRET in C. elegans illuminates functional receptor interactions in real time

BackgroundProtein-protein interactions form the basis of every organism and thus, investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligand-binding are of general interest. Bioluminescence resonance energy transfer (BRET) is a powerful tool to investigate these aspects in vitro. Since in vitro approaches mostly neglect the more complex in vivo situation, we established BRET as an in vivo tool for studying protein interactions in the nematode C. elegans.ResultsWe generated worms expressing NanoBRET sensors and elucidated the interaction of two ligand-G protein-coupled receptor (GPCR) pairs, the neuropeptide receptor NPR-11 and the Adhesion GPCR LAT-1. Furthermore, we adapted the enhanced bystander BRET technology to measure subcellular protein localization. Using this approach, we traced ligand-induced internalization of NPR-11 in vivo.ConclusionsOur results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. elegans.

PSL-LCCL: a resource for subcellular protein localization in liver cancer cell line SK_HEP1.

The characterization of subcellular protein localization provides a basis for further understanding cellular behaviors. A delineation of subcellular localization of proteins on cytosolic membrane-bound organelles in human liver cancer cell lines (hLCCLs) has yet to be performed. To obtain its proteome-wide view, we isolated and enriched six cytosolic membrane-bound organelles in one of the hLCCLs (SK_HEP1) and quantified their proteins using mass spectrometry. The vigorous selection of marker proteins and a machine-learning-based algorithm were implemented to localize proteins at cluster and neighborhood levels. We validated the performance of the proposed method by comparing the predicted subcellular protein localization with publicly available resources. The profiles enabled investigating the correlation of protein domains with their subcellular localization and colocalization of protein complex members. A subcellular proteome database for SK_HEP1, including (i) the subcellular protein localization and (ii) the subcellular locations of protein complex members and their interactions, was constructed. Our research provides resources for further research on hLCCLs proteomics. Database URL: http://www.igenetics.org.cn/project/PSL-LCCL/

Deep localization of subcellular protein structures from fluorescence microscopy images

Deep localization of subcellular protein structures from fluorescence microscopy images

Is autophagy induction by PARP inhibitors a target for therapeutic benefit?

PARP inhibitors have proven to be effective in conjunction with conventional therapeutics in the treatment of various solid as well as hematologic malignancies, particularly when the tumors are deficient in DNA repair pathways. However, as the case with other chemotherapeutic agents, their effectiveness is often compromised by the development of resistance. PARP inhibitors have consistently been reported to promote autophagy, a process that maintains cellular homeostasis and acts as an energy source by the degradation and reutilization of damaged subcellular organelles and proteins. Autophagy can exhibit different functional properties, the most prominent being cytoprotective. In addition, both cytotoxic and non-protective functions forms have also been identified. In this review, we explore the available literature regarding the different roles of autophagy in response to clinically-used PARP inhibitors, highlighting the possibility of targeting autophagy as an adjuvant therapy to potentially increase the effectiveness of PARP inhibition and to overcome the development of resistance.

Integrated Network Discovery Using Multi-Proteomic Data.

A fundamental goal of systems biology is to seek a better understanding of the cell's molecular mechanisms. Experimentalists most frequently rely upon reductionist methods to isolate and analyze discrete signaling compartments, including subcellular domains, organelles, and protein-protein interactions. Among the systems-biology community, there is a growing need to integrate multiple datasets to resolve complex cellular networks. In this chapter, we share our procedures for the discovery of integrated signaling networks, across multi-proteomic data. Demonstrating these procedures, we provide an integrated analysis of the cellular proteome and extracellular (secretome) of human glioma LN229.

Proteomic Mapping by APEX2-Catalyzed Proximity Labeling in Saccharomyces cerevisiae Semipermeabilized Cells.

Enzyme-catalyzed proximity labeling (PL) has proven to be a valuable resource for proteomic mapping of subcellular compartments and protein networks in living cells. We have used engineered ascorbate peroxidase (APEX2) to develop a PL approach for budding yeast. It is based on semipermeabilized cells to overcome poor cellular permeability of the APEX2 substrate biotin-phenol and difficulties in its delivery into the cell. The use of semipermeabilized cells has several advantages, in particular the avoidance of generating fragile spheroplasts and the opportunity of employing cells from a glucose-containing medium for APEX2 tagging. In this protocol we describe how to perform a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach that allows to map an open cellular compartment like the yeast nucleus. In particular, we focus on the proteomic sample preparation and provide instructions to achieve high-resolution mapping of a subcellular yeast proteome.

Digital Imaging Analysis for Quantitative Subcellular Localization of MRP2 during Hepatic Ischemia–Reperfusion Injury in a Rat Model: Can You Capture Time?

Introduction: The mechanism of cholestasis in hepatic ischemia–reperfusion injury (IRI) remains to be elucidated. Short-term changes in bile formation are regulated by translocating transporter proteins between the cell membrane and the cytosolic compartment. We sought to develop a method that quantifies the degree of translocation of multidrug resistance-associated protein 2 (MRP2) along the hepatocyte canalicular membrane. Method: Fifteen male Sprague Dawley rats were divided into three groups based on warm ischemia time (WIT): 0 min, 10 min, and 20 min. IRI was induced by temporarily clamping pedicles to the median and lateral lobes, followed by 60 min of reperfusion. Liver tissue was analyzed for subcellular protein fractionation and immunofluorescence staining. CD13 served as a reference protein for the canalicular membrane. Digital imaging analyses measured the area of MRP2 and CD13 staining. The size of the MRP2 area without overlap with CD13 (i.e., MRP2 in the cytosolic compartment) in proportion to the total MRP2 area was defined as the MRP2 translocation index (MTI). Results: The proportion of CD13 along the canalicular membrane was larger than that of MRP2 (median 93.3% versus 43.4%, respectively; P<0.0001, Figure A). The subcellular distribution of CD13 was consistent among the three WIT groups. Immunofluorescence colocalization analysis for CD13 and MRP2 demonstrated a negative linear correlation between MTI and WIT (P=0.0183, Y=-1.052X+25.00, Figure B). Conclusions: The degree of MRP2 translocation relative to CD13 was quantified, which correlated with WIT. This novel approach can be used to determine the pathophysiology of cholestasis in hepatic IRI.

Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons.

Over the past years several forms of superresolution fluorescence microscopy have been developed that offer the possibility to study cellular structures and protein distribution at a resolution well below the diffraction limit of conventional fluorescence microscopy (<200nm). A particularly powerful superresolution technique is single-molecule localization microscopy (SMLM). SMLM enables the quantitative investigation of subcellular protein distribution at a spatial resolution up to tenfold higher than conventional imaging, even in live cells. Not surprisingly, SMLM has therefore been used in many applications in biology, including neuroscience. This chapter provides a step-by-step SMLM protocol to visualize the nanoscale organization of endogenous proteins in dissociated neurons but can be extended to image other adherent cultured cells. We outline a number of methods to visualize endogenous proteins in neurons for live-cell and fixed application, including immunolabeling, the use of intrabodies for live-cell SMLM, and endogenous tagging using CRISPR/Cas9.

Development of destabilized mCherry fluorescent proteins for applications in the model yeast Saccharomyces cerevisiae

Fluorescent proteins are widely used molecular reporters in studying gene expression and subcellular protein localization. To enable the monitoring of transient cellular events in the model yeast Saccharomyces cerevisiae, destabilized green and cyan fluorescent proteins have been constructed. However, their co-utilization is limited by an overlap in their excitation and emission spectra. Although red fluorescent protein is compatible with both green and cyan fluorescent proteins with respect to spectra resolution, a destabilized red fluorescent protein is yet to be constructed for applications in S. cerevisiae. To realize this, we adopted a degron-fusion strategy to prompt destabilization of red fluorescent protein. Specifically, we fused two degrons derived from Cln2, a G1-specific cyclin that mediates cell cycle transition, to the N- or C-terminus of mCherry to generate four destabilized fluorescent proteins that are soluble and functional in S. cerevisiae. Importantly, the four mCherry fluorescent proteins are highly differential with regards to fluorescence half-life and intensity, which provides a greater choice of tools available for the study of dynamic gene expression and transient cellular processes in the model yeast.

Translation-dependent mRNA localization to Caenorhabditis elegans adherens junctions.

ABSTRACTmRNA localization is an evolutionarily widespread phenomenon that can facilitate subcellular protein targeting. Extensive work has focused on mRNA targeting through ‘zip-codes’ within untranslated regions (UTRs), whereas much less is known about translation-dependent cues. Here, we examine mRNA localization in Caenorhabditis elegans embryonic epithelia. From an smFISH-based survey, we identified mRNAs associated with the cell membrane or cortex, and with apical junctions in a stage- and cell type-specific manner. Mutational analyses for one of these transcripts, dlg-1/discs large, revealed that it relied on a translation-dependent process and did not require its 5′ or 3′ UTRs. We suggest a model in which dlg-1 transcripts are co-translationally localized with the nascent protein: first the translating complex goes to the cell membrane using sequences located at the C-terminal/3′ end, and then apically using N-terminal/5′ sequences. These studies identify a translation-based process for mRNA localization within developing epithelia and determine the necessary cis-acting sequences for dlg-1 mRNA targeting.