A comparative urinary proteomic and metabolomic analysis between renal aa amyloidosis and membranous nephropathy with clinicopathologic correlations.

Urinary omics has become a powerful tool for elucidating pathophysiology of glomerular diseases. However, no urinary omics analysis has been performed yet on renal AA amyloidosis. Here, we performed a comparative urine proteomic and metabolomic analysis between recently diagnosed renal AA amyloidosis (AA) and membranous nephropathy (MN) patients. Urine samples of 22 (8 AA, 8 MN and 6 healthy control) patients were analyzed with nLC-MS/MS and GC/MS for proteomic and metabolomic studies, respectively. Pathological specimens were scored for glomerulosclerosis and tubulointerstitial fibrosis grades. Functional enrichment analysis between AA and control groups showed enrichment in cell adhesion related sub-domains. Uromodulin (UMOD) was lower, whereas ribonuclease 1 (RNase1) and α-1-microglobulin/bikunin precursor (AMBP) were higher in AA compared to MN group. Correlations were demonstrated between UMOD-proteinuria (r = -0.48, p = 0.03) and AMBP-eGFR (r = -0.69, p = 0.003) variables. Metabolomic analysis showed myo-inositol and urate were higher in AA compared to MN group. A positive correlation was detected between RNase1 and urate independent of eGFR values (r = 0.63, p = 0.01). Enrichment in cell adhesion related domains suggested a possible increased urinary shear stress due to amyloid fibrils. UMOD, AMBP and myo-inositol were related with tubulointerstitial damage, whereas RNase1 and urate were believed to be related with systemic inflammation in AA amyloidosis. SIGNIFICANCE: Urinary omics studies have become a standard tool for biomarker studies. However, no urinary omics analysis has been performed yet on renal AA amyloidosis. Here, we performed a comparative urinary omics analysis between recently diagnosed renal AA amyloidosis (AA), membranous nephropathy (MN) patients and healthy controls. Pathological specimens were scored with glomerulosclerosis (G) and tubulointerstitial fibrosis (IF) grades to consolidate the results of the omics studies and correlation analyzes. Functional enrichment analysis showed enrichment in cell adhesion related sub-domains due to downregulation of cadherins; which could be related with increased urinary shear stress due to amyloid deposition and disruption of tissue micro-architecture. In comparative proteomic analyzes UMOD was lower, whereas RNase1 and AMBP were higher in AA compared to MN group. Whereas in metabolomic analyzes; myo-inositol, urate and maltose were higher in AA compared to MN group. Correlations were demonstrated between UMOD-proteinuria (r = -0.48, p = 0.03), AMBP-eGFR (r = -0.69, p = 0.003) and between RNase1-Urate independent of eGFR values (r = 0.63, p = 0.01). This study is the first comprehensive urinary omics analysis focusing on renal AA Amyloidosis to the best of our knowledge. Based on physiologic roles and clinicopathologic correlations of the molecules; UMOD, AMBP and myo-inositol were related with tubulointerstitial damage, whereas RNase1 and urate were believed to be increased with systemic inflammation and endothelial damage in AA amyloidosis.

TW68, cryptochromes stabilizer, regulates fasting blood glucose levels in diabetic ob/ob and high fat-diet-induced obese mice.

Cryptochromes (CRYs), transcriptional repressors of the circadian clock in mammals, inhibit cAMP production when glucagon activates G-protein coupled receptors. Therefore, molecules that modulate CRYs have the potential to regulate gluconeogenesis. In this study, we discovered a new molecule called TW68 that interacts with the primary pockets of mammalian CRY1/2, leading to reduced ubiquitination levels and increased stability. In cell-based circadian rhythm assays using U2OS Bmal1-dLuc cells, TW68 extended the period length of the circadian rhythm. Additionally, TW68 decreased the transcriptional levels of two genes, Phosphoenolpyruvate carboxykinase 1 (PCK1) and Glucose-6-phosphatase (G6PC), which play crucial roles in glucose biosynthesis during glucagon-induced gluconeogenesis in HepG2 cells. Oral administration of TW68 in mice showed good tolerance, a good pharmacokinetic profile, and remarkable bioavailability. Finally, when administered to fasting diabetic animals from ob/ob and HFD-fed obese mice, TW68 reduced blood glucose levels by enhancing CRY stabilization and subsequently decreasing the transcriptional levels of Pck1 and G6pc. These findings collectively demonstrate the antidiabetic efficacy of TW68 in vivo, suggesting its therapeutic potential for controlling fasting glucose levels in the treatment of type 2 diabetes mellitus.

Cell cycle-dependent palmitoylation of protocadherin 7 by ZDHHC5 promotes successful cytokinesis.

Cell division requires dramatic reorganization of the cell cortex, which is primarily driven by the actomyosin network. We previously reported that protocadherin 7 (PCDH7) gets enriched at the cell surface during mitosis, which is required to build up the full mitotic rounding pressure. Here, we report that PCDH7 interacts with and is palmitoylated by the palmitoyltransferase, ZDHHC5. PCDH7 and ZDHHC5 colocalize at the mitotic cell surface and translocate to the cleavage furrow during cytokinesis. The localization of PCDH7 depends on the palmitoylation activity of ZDHHC5. Silencing PCDH7 increases the percentage of multinucleated cells and the duration of mitosis. Loss of PCDH7 expression correlates with reduced levels of active RhoA and phospho-myosin at the cleavage furrow. This work uncovers a palmitoylation-dependent translocation mechanism for PCDH7, which contributes to the reorganization of the cortical cytoskeleton during cell division.

Plasma proteomics identify potential severity biomarkers from COVID-19 associated network.

PURPOSE: Coronavirus disease 2019 (COVID-19) continues to threaten public health globally. Severe acute respiratory coronavirus type 2 (SARS-CoV-2) infection-dependent alterations in the host cell signaling network may unveil potential target proteins and pathways for therapeutic strategies. In this study, we aim to define early severity biomarkers and monitor altered pathways in the course of SARS-CoV-2 infection. EXPERIMENTAL DESIGN: We systematically analyzed plasma proteomes of COVID-19 patients from Turkey by using mass spectrometry. Different severity grades (moderate, severe, and critical) and periods of disease (early, inflammatory, and recovery) are monitored. Significant alterations in protein expressions are used to reconstruct the COVID-19 associated network that was further extended to connect viral and host proteins. RESULTS: Across all COVID-19 patients, 111 differentially expressed proteins were found, of which 28 proteins were unique to our study mainly enriching in immunoglobulin production. By monitoring different severity grades and periods of disease, CLEC3B, MST1, and ITIH2 were identified as potential early predictors of COVID-19 severity. Most importantly, we extended the COVID-19 associated network with viral proteins and showed the connectedness of viral proteins with human proteins. The most connected viral protein ORF8, which has a role in immune evasion, targets many host proteins tightly connected to the deregulated human plasma proteins. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma proteomes from critical patients are intrinsically clustered in a distinct group than severe and moderate patients. Importantly, we did not recover any grouping based on the infection period, suggesting their distinct proteome even in the recovery phase. The new potential early severity markers can be further studied for their value in the clinics to monitor COVID-19 prognosis. Beyond the list of plasma proteins, our disease-associated network unravels altered pathways, and the possible therapeutic targets in SARS-CoV-2 infection by connecting human and viral proteins. Follow-up studies on the disease associated network that we propose here will be useful to determine molecular details of viral perturbation and to address how the infection affects human physiology.

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice.

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.

Quantitative Phosphoproteomics Analysis Uncovers PAK2- and CDK1-Mediated Malignant Signaling Pathways in Clear Cell Renal Cell Carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is among the 10 most common cancers in both men and women and causes more than 140,000 deaths worldwide every year. In order to elucidate the underlying molecular mechanisms orchestrated by phosphorylation modifications, we performed a comprehensive quantitative phosphoproteomics characterization of ccRCC tumor and normal adjacent tissues. Here, we identified 16,253 phosphopeptides, of which more than 9000 were singly quantified. Our in-depth analysis revealed 600 phosphopeptides to be significantly differentially regulated between tumor and normal tissues. Moreover, our data revealed that significantly up-regulated phosphoproteins are associated with protein synthesis and cytoskeletal re-organization which suggests proliferative and migratory behavior of renal tumors. This is supported by a mesenchymal profile of ccRCC phosphorylation events. Our rigorous characterization of the renal phosphoproteome also suggests that both epidermal growth factor receptor and vascular endothelial growth factor receptor are important mediators of phospho signaling in RCC pathogenesis. Furthermore, we determined the kinases p21-activated kinase 2, cyclin-dependent kinase 1 and c-Jun N-terminal kinase 1 to be master kinases that are responsible for phosphorylation of many substrates associated with cell proliferation, inflammation and migration. Moreover, high expression of p21-activated kinase 2 is associated with worse survival outcome of ccRCC patients. These master kinases are targetable by inhibitory drugs such as fostamatinib, minocycline, tamoxifen and bosutinib which can serve as novel therapeutic agents for ccRCC treatment.

Proteome analysis of the circadian clock protein PERIOD2.

Circadian rhythms are a series of endogenous autonomous 24-h oscillations generated by the circadian clock. At the molecular level, the circadian clock is based on a transcription-translation feedback loop, in which BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME (CRY) and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. There are three PER proteins, of which PER2 shows the strongest oscillation at both stability and cellular localization level. Protein-protein interactions (PPIs) or interactome of the circadian clock proteins have been investigated using classical methods such as two-dimensional gel electrophoresis, immunoprecipitation-coupled mass spectrometry, and yeast-two hybrid assay where the dynamic and weak interactions are difficult to catch. To identify the interactome of PER2 we have adopted proximity-dependent labeling with biotin and mass spectrometry-based identification of labeled proteins (BioID). In addition to known interactions with such as CRY1 and CRY2, we have identified several new PPIs for PER2 and confirmed some of them using co-immunoprecipitation technique. This study characterizes the PER2 protein interactions in depth, and it also implies that using a fast BioID method with miniTurbo or TurboID coupled to other major circadian clock proteins might uncover other interactors in the clock that have yet to be discovered.

Adaptive tracking algorithm for trajectory analysis of cells and layer-by-layer assessment of motility dynamics.

Tracking biological objects such as cells or subcellular components imaged with time-lapse microscopy enables us to understand the molecular principles about the dynamics of cell behaviors. However, automatic object detection, segmentation and extracting trajectories remain as a rate-limiting step due to intrinsic challenges of video processing. This paper presents an adaptive tracking algorithm (Adtari) that automatically finds the optimum search radius and cell linkages to determine trajectories in consecutive frames. A critical assumption in most tracking studies is that displacement remains unchanged throughout the movie and cells in a few frames are usually analyzed to determine its magnitude. Tracking errors and inaccurate association of cells may occur if the user does not correctly evaluate the value or prior knowledge is not present on cell movement. The key novelty of our method is that minimum intercellular distance and maximum displacement of cells between frames are dynamically computed and used to determine the threshold distance. Since the space between cells is highly variable in a given frame, our software recursively alters the magnitude to determine all plausible matches in the trajectory analysis. Our method therefore eliminates a major preprocessing step where a constant distance was used to determine the neighbor cells in tracking methods. Cells having multiple overlaps and splitting events were further evaluated by using the shape attributes including perimeter, area, ellipticity and distance. The features were applied to determine the closest matches by minimizing the difference in their magnitudes. Finally, reporting section of our software were used to generate instant maps by overlaying cell features and trajectories. Adtari was validated by using videos with variable signal-to-noise, contrast ratio and cell density. We compared the adaptive tracking with constant distance and other methods to evaluate performance and its efficiency. Our algorithm yields reduced mismatch ratio, increased ratio of whole cell track, higher frame tracking efficiency and allows layer-by-layer assessment of motility to characterize single-cells. Adaptive tracking provides a reliable, accurate, time efficient and user-friendly open source software that is well suited for analysis of 2D fluorescence microscopy video datasets.

Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation.

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation.

BACKGROUND: The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. RESULTS: We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. CONCLUSIONS: Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.

Structural analysis of mammalian protein phosphorylation at a proteome level.

Phosphorylation is an essential post-translational modification for almost all cellular processes. Several global phosphoproteomics analyses have revealed phosphorylation profiles under different conditions. Beyond identification of phospho-sites, protein structures add another layer of information about their functionality. In this study, we systematically characterize phospho-sites based on their 3D locations in the protein and establish a location map for phospho-sites. More than 250,000 phospho-sites have been analyzed, of which 8,686 sites match at least one structure and are stratified based on their respective 3D positions. Core phospho-sites possess two distinct groups based on their dynamicity. Dynamic core phosphorylations are significantly more functional compared with static ones. The dynamic core and the interface phospho-sites are the most functional among all 3D phosphorylation groups. Our analysis provides global characterization and stratification of phospho-sites from a structural perspective that can be utilized for predicting functional relevance and filtering out false positives in phosphoproteomic studies.

Quantitative Proteomics Identifies Secreted Diagnostic Biomarkers as well as Tumor-Dependent Prognostic Targets for Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the third most common and most malignant urological cancer, with a 5-year survival rate of 10% for patients with advanced tumors. Here, we identified 10,160 unique proteins by in-depth quantitative proteomics, of which 955 proteins were significantly regulated between tumor and normal adjacent tissues. We verified four putatively secreted biomarker candidates, namely, PLOD2, FERMT3, SPARC, and SIRPα, as highly expressed proteins that are not affected by intratumor and intertumor heterogeneity. Moreover, SPARC displayed a significant increase in urine samples of patients with ccRCC, making it a promising marker for the detection of the disease in body fluids. Furthermore, based on molecular expression profiles, we propose a biomarker panel for the robust classification of ccRCC tumors into two main clusters, which significantly differed in patient outcome with an almost three times higher risk of death for cluster 1 tumors compared with cluster 2 tumors. Moreover, among the most significant clustering proteins, 13 were targets of repurposed inhibitory FDA-approved drugs. Our rigorous proteomics approach identified promising diagnostic and tumor-discriminative biomarker candidates which can serve as therapeutic targets for the treatment of ccRCC. IMPLICATIONS: Our in-depth quantitative proteomics analysis of ccRCC tissues identifies the putatively secreted protein SPARC as a promising urine biomarker and reveals two molecular tumor phenotypes.

Proteome profiling of neuron-derived exosomes in Alzheimer's disease reveals hemoglobin as a potential biomarker.

Alzheimer's disease is a chronic and progressive neurodegenerative disorder, which is the most common cause of dementia worldwide. Although amyloid plaques and neurofibrillary tangles are identified as the hallmarks of the disease, the only valid diagnostic method yet is post-mortem imaging of these molecules in brain sections. Exosome is a type of extracellular vesicles secreted into extracellular space and plays fundamental roles in healthy and pathological conditions, including cell-to-cell communication. In this study, we aimed to investigate the proteomic contents of neuron-derived exosomes (NDEs) from AD patients and healthy controls (HCs) to identify a possible marker for AD diagnosis. We identified alpha-globin, beta-globin, and delta-globin increase in neuron-derived exosomes of AD patients compared to HCs with LC-MS/MS proteomics analysis. Then, we confirmed the high hemoglobin (Hb) level in NDEs of AD patients with ELISA. We found the area under the curve of hemoglobin level as 0.6913 with ROC analysis. Cargo proteins of NDEs may be useful diagnostic biomarker for AD.

Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation.

KIF2A is a kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. However, how different KIF2A alternative isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in the developing cortex. We show that Kif2a is essential for dendritic arborization in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons; a third isoform, lacking a key N-terminal region, is ineffective. By proximity-based interactome mapping for individual isoforms, we identify previously known KIF2A interactors, proteins localized to the mitotic spindle poles and, unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to organelles, prominently to the mitochondria. In addition, we show that a KIF2A mutation, which causes brain malformations in humans, has extensive changes to its proximity-based interactome, with depletion of mitochondrial proteins identified in the wild-type KIF2A interactome. Our data raises new insights about the importance of alternative splice variants during brain development.

Proximal Biotinylation-Based Combinatory Approach for Isolating Integral Plasma Membrane Proteins.

Comprehensive profiling of the cell-surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step, which we call Plasma Membrane-BioID. The lipid-modified BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of the plasma membrane, where it effectively biotinylated plasma membrane proteins. Biotinylated proteins were then affinity-purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and Plasma Membrane-BioID is ideal to overcome the inherent limitations of the identification of integral membrane proteins, and it yields highly pure plasma components for downstream proteomic analysis.

CLIC4 and CLIC1 bridge plasma membrane and cortical actin network for a successful cytokinesis.

CLIC4 and CLIC1 are members of the well-conserved chloride intracellular channel proteins (CLICs) structurally related to glutathione-S-transferases. Here, we report new roles of CLICs in cytokinesis. At the onset of cytokinesis, CLIC4 accumulates at the cleavage furrow and later localizes to the midbody in a RhoA-dependent manner. The cell cycle-dependent localization of CLIC4 is abolished when its glutathione S-transferase activity-related residues (C35A and F37D) are mutated. Ezrin, anillin, and ALIX are identified as interaction partners of CLIC4 at the cleavage furrow and midbody. Strikingly, CLIC4 facilitates the activation of ezrin at the cleavage furrow and reciprocally inhibition of ezrin activation diminishes the translocation of CLIC4 to the cleavage furrow. Furthermore, knockouts of CLIC4 and CLIC1 cause abnormal blebbing at the polar cortex and regression of the cleavage furrow at late cytokinesis leading to multinucleated cells. We conclude that CLIC4 and CLIC1 function together with ezrin where they bridge plasma membrane and actin cytoskeleton at the polar cortex and cleavage furrow to promote cortical stability and successful completion of cytokinesis in mammalian cells.

Terminal neuron localization to the upper cortical plate is controlled by the transcription factor NEUROD2.

Excitatory neurons of the mammalian cerebral cortex are organized into six functional layers characterized by unique patterns of connectivity, as well as distinctive physiological and morphological properties. Cortical layers appear after a highly regulated migration process in which cells move from the deeper, proliferative zone toward the superficial layers. Importantly, defects in this radial migration process have been implicated in neurodevelopmental and psychiatric diseases. Here we report that during the final stages of migration, transcription factor Neurogenic Differentiation 2 (Neurod2) contributes to terminal cellular localization within the cortical plate. In mice, in utero knockdown of Neurod2 resulted in reduced numbers of neurons localized to the uppermost region of the developing cortex, also termed the primitive cortical zone. Our ChIP-Seq and RNA-Seq analyses of genes regulated by NEUROD2 in the developing cortex identified a number of key target genes with known roles in Reelin signaling, a critical regulator of neuronal migration. Our focused analysis of regulation of the Reln gene, encoding the extracellular ligand REELIN, uncovered NEUROD2 binding to conserved E-box elements in multiple introns. Furthermore, we demonstrate that knockdown of NEUROD2 in primary cortical neurons resulted in a strong increase in Reln gene expression at the mRNA level, as well as a slight upregulation at the protein level. These data reveal a new role for NEUROD2 during the late stages of neuronal migration, and our analysis of its genomic targets offers new genes with potential roles in cortical lamination.

Systems-level Analysis Reveals Multiple Modulators of Epithelial-mesenchymal Transition and Identifies DNAJB4 and CD81 as Novel Metastasis Inducers in Breast Cancer.

Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared with the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased on EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistance.

Extracellular glucose level regulates dependence on GRP78 for cell surface localization of multipass transmembrane proteins in HeLa cells.

Many human-cultured cell lines survive glucose starvation, but the underlying mechanisms remain unclear. Here, we searched for proteins required for cellular adaptation to glucose-limited conditions and identified several endoplasmic reticulum chaperones in the glucose-regulated protein (GRP) family as proteins enriched in the cellular membrane. Surprisingly, these proteins, which are required for cell surface localization of GLUT1 under high-glucose conditions, become dispensable for targeting GLUT1 to the surface upon glucose starvation. In marked contrast, cell surface localization of single-pass transmembrane proteins, such as epidermal growth factor receptor and CD98, is not disturbed by GRP78 depletion regardless of the extracellular glucose level. These results indicate that the extracellular glucose level regulates dependence on the GRPs for cell surface localization of multipass transmembrane proteins.

Labeling Carboxyl Groups of Surface-Exposed Proteins Provides an Orthogonal Approach for Cell Surface Isolation.

Quantitative profiling of cell surface proteins is critically important for the understanding of cell-cell communication, signaling, tissue development, and homeostasis. Traditional proteomics methods are challenging for cell surface proteins due to their hydrophobic nature and low abundance, necessitating alternative methods to efficiently identify and quantify this protein group. Here we established carboxyl-reactive biotinylation for selective and efficient biotinylation and isolation of surface-exposed proteins of living cells. We assessed the efficiency of carboxyl-reactive biotinylation for plasma membrane proteins by comparing it with a well-established protocol, amine-reactive biotinylation, using SILAC (stable isotope labeling in cell culture). Our results show that carboxyl-reactive biotinylation of cell surface proteins is both more selective and more efficient than amine-reactive biotinylation. We conclude that it is a useful approach, which is partially orthogonal to amine-reactive biotinylation, allowing us to cast a wider net for a comprehensive profiling of cell surface proteins.