Membrane Proteome Research Articles

A comparison of milk fat globule membranes and whey proteomes: New insight into variation nutrient differences between Buffalo, Cow, Goat, and Yak.

In this study, the whey and milk fat globule membrane (MFGM) proteomes of buffalo, cow, goat, and yak milk were analyzed using label-free proteomic technology. Totally, 1,292 MFGM proteins and 686 whey proteins were identified from these four species, and GO analysis revealed there were specific proteins with different functions in both whey (376) and MFGM (982) proteomes. The principal component analysis showed that ALB, TF, CSN1S1, and GLYCAM1 are characteristic markers of the milk for each of the four species. Furthermore, the conserved and differential in the expression of whey and MFGM proteins across the four species were identified by limma, and subsequent KEGG pathway analysis showed that immune-related proteins are both conserved and species-specific in the four species. These results provide a deepening of the understanding of the characteristics of proteins in whey and MFGMs from these four common dairy animals and new insight into developing dairy production.

Urea Transporter B Is Produced in the Jk Null Blood Group Yet Fails to Integrate into the Red Cell Membrane Impacting Erythropoiesis and Erythrocytes

The Kidd (Jk) blood group is carried by an integral membrane glycoprotein, urea transporter B (UT-B), which transports urea through the red blood cell (RBC) membrane. The absence of both Jk a and Jk b antigens, designated phenotypically as Jk(a-b-) (Jk null), is very rare with a significantly increased prevalence in certain ethnicities, most abundantly within the Polynesian population where it is found at 0.9% prevalence. Provision of blood for patients requiring this phenotype is challenging. In Australia, there are only 15 active Jk null blood donors. Individuals of the Jk null phenotype have been shown to have suboptimal urine concentrating ability but erythrocytes appear phenotypically normal with shape, size and lifespan within normal reference indices (Lawicki et al., Transfus Med Rev, 2017). Where UT-B integrates in the RBC membrane is unknown although protein 4.1R deficiency is reported to lead to the absence of UT-B (Azouzi et al., Br J Haematol, 2015). Our aim was to identify and characterise the molecular basis for the Jk null phenotype in Australian blood donors and decipher the biological impact of the Jk null phenotype using our ex vivo model of erythropoiesis to help inform clinical transfusion practice. Massively parallel sequencing showed that all blood donors (n=9) were homozygous for JK c.342-1G>A (splice site variant) and c.838G>A polymorphisms resulting in the JK*02N.01/*02N.01 genotype. This genotype predicts a Jk null phenotype, and a truncated UT-B protein caused by the formation of a premature stop codon. The assumption from the genomics data is that the putative truncated UT-B protein would not be translated. To test this hypothesis we examined the RBCs of Jk null individuals and confirmed the lack of UT-B expression on the RBC surface by live flow cytometry and confocal imaging. In control RBCs UT-B is seen in discrete microdomains on the RBC surface by live confocal microscopy. To establish the expression of UT-B during erythropoiesis we cultured CD34+ cells from control and Jk null buffy coats after whole blood donation. Flow cytometry and confocal microscopy of control cells identified that UT-B is expressed intracellularly and at the cell surface in the developing erythroid cells. Notably we identified that the UT-B protein can also be found intracellularly in developing Jk null erythroid cells but is not detectable in the plasma membrane. In Jk null reticulocytes, UT-B colocalises with markers of protein degradation and destruction implying that the truncated UT-B protein is quickly removed by controlled protein degradation pathways. Quantitative proteomics was used to assess the global effect of the Jk null phenotype on the mature RBC membrane proteome. These data confirmed the absence of UT-B but also uncovered that in mature Jk null RBCs, RBC proteins involved in solute transport (aquaporin-1, stomatin) and membrane-cytoskeleton stabilisation (alpha and beta adducins, and dematin) are significantly downregulated. In contrast, actin binding proteins including tropomyosin and myosin IIA were upregulated in Jk null RBCs. The proteomics data also revealed that numerous major plasma proteins including alpha-2-macroglobulin, hemopexin and immunoglobulins are associated exclusively with Jk null RBCs. These findings imply that the lack of functional, membrane embedded UT-B induces various compensatory mechanisms in the Jk null RBCs membrane. In conclusion, we show that a truncated UT-B protein is produced in Jk null erythroid cells but fails to mature into a RBC membrane integrated, functional glycoprotein. Our data also suggest that the absence of a functional UT-B glycoprotein on the RBC surface affects the RBC membrane and cytoskeletal proteome and its interaction with major plasma glycoproteins.

Identification of Surface Markers and Functional Characterization of Myeloid Derived Suppressor Cell-Like Adherent Cells.

Myeloid-derived suppressor cell (MDSC)-like adherent cells (MLACs) are a recently identified CD11b+ F4/80- myeloid cell subset that can infiltrate tumors early in development and promote their growth. Because of these functions, MLACs play an important role in establishing an immunosuppressive tumor microenvironment (TME). However, the lack of MLAC-specific markers has hampered further characterization of this cell type. This study identifies the gene signature of MLACs by analyzing RNA-sequencing (RNA-seq) and public single-cell RNA-seq data, revealing that MLACs are an independent cell population that are distinct from other intratumoral myeloid cells. After combining proteome analysis of membrane proteins with RNA-seq data, H2-Ab1 and CD11c are indicated as marker proteins that can support the isolation of MLAC subsets from CD11b+ F4/80- myeloid cells by fluorescence-activated cell sorting. The CD11b+ F4/80- H2-Ab1+ and CD11b+ F4/80- CD11c+ MLAC subsets represent approximately half of the MLAC population that is isolated based on their adhesion properties and possess gene signatures and functional properties similar to those of the MLAC population. Additionally, membrane proteome analysis suggests that MLACs express highly heterogeneous surface proteins. This study facilitates an integrated understanding of heterogeneous intratumoral myeloid cells, as well as the molecular and cellular details of the development of an immunosuppressive TME.

Escherichia coli displays a conserved membrane proteomic response to a range of alcohols

BackgroundAlcohol is a good and environment-friendly fuel that can be microbially produced, capable of eliminating many of the limitations of the present-day fossil fuels. However, the inherent toxic nature of alcohols to the microbial cells leads to end-product inhibition that limits large-scale alcohol production by fermentation. Fundamental knowledge about the stress responses of microorganisms to alcohols would greatly facilitate to improve the microbial alcohol tolerance. The current study elucidates and compares the changes in the membrane proteome of Escherichia coli in response to a range of alcohols.ResultsAlthough alcohol toxicity increased exponentially with alcohol chain length (2–6 carbon), similar stress responses were observed in the inner and outer membrane proteome of E. coli in the presence of 2-, 4- and 6-carbon alcohols at the MIC50. This pertains to: (1) increased levels of inner membrane transporters for uptake of energy-producing metabolites, (2) reduced levels of non-essential proteins, associated with anaerobic, carbon starvation and osmotic stress, for energy conservation, (3) increased levels of murein degrading enzymes (MltA, EmtA, MliC and DigH) promoting cell elongation and 4) reduced levels of most outer membrane β-barrel proteins (LptD, FadL, LamB, TolC and BamA). Major outer membrane β-barrel protein OmpC, which is known to contribute to ethanol tolerance and membrane integrity, was notably reduced by alcohol stress. While LPS is important for OmpC trimerisation, LPS release by EDTA did not lower OmpC levels. This suggests that LPS release, which is reported under alcohol stress, does not contribute to the reduced levels of OmpC in the presence of alcohol.ConclusionsSince alcohol primarily targets the integrity of the membrane, maintenance of outer membrane OmpC levels in the presence of alcohol might help in the survival of E. coli to higher alcohol concentrations. The study provides important information about the membrane protein responses of E. coli to a range of alcohols, which can be used to develop targeted strategies for increased microbial alcohol tolerance and hence bioalcohol production.

Subinhibitory Levels of Fluoroquinolones Result in Enrichment of the Membrane Proteome of Staphylococcus aureus

Subinhibitory Levels of Fluoroquinolones Result in Enrichment of the Membrane Proteome of Staphylococcus aureus

DIFFERENTIAL CELL SURFACE PROTEOMICS OF FLOTILLIN-1, HMGB1 AND AMINOPEPTIDASE IN H3 MUTATED PAEDIATRIC HIGH-GRADE GLIOMA

Abstract AIMS High-grade gliomas (HGG) are the deadliest central nervous system tumour in children. Despite this, treatment options remain elusive, leaving children with a dismal prognosis. Histone H3.3 mutations, K27M and G34V/R have been identified as the two most common recurrent somatic mutations in paediatric HGG (pHGG). These mutations are present in 50% of pHGG compared to only 1% in adults. Our previous research on cell membrane protein analyses have identified different signature of unique and common cell membrane proteins in H3-G34R/V mutated pHGG, in comparison to H3 wild type tumour. Among these we have identified flotillin-1, HMGB1 and aminopeptidase N. Flotillin-1 promotes numerous malignancies, High mobility group box 1(HMGB1) functions as an oncogene and aminopeptidase N is a cell surface metallo- protease involved in tumour progression. We have now verified the expression and localization of these cell surface proteins in H3 mutated and wildtype tumour, in order to target them for developing novel therapeutics. METHOD Cell membrane proteomics were further verified though mass-spectrometric data analyses. Expression and localization of flotillin-1, HMGB1 and Aminopeptidase in H3.3 mutated pHGG cell lines in comparison to H3 wildtype glioma were analysed, using different protein expression assays including immunofluorescence microscopy and western blot assays. RESULTS Experimental analyses showed differential protein expression and localization of Flotillin-1, HMGB1 and Aminopeptidase and verified the mass-spectrometric proteomic data in H3-G34V pHGG, in comparison to wild- type pHGG. CONCLUSIONS This new knowledge will help us utilise Flotillin-1, HMGB1 and Aminopeptidase N, as potential therapeutic targets for these unique H3 mutated children’s brain tumours.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8h post-infusion and remained at the site for over 10days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.

SYST-07 PLASMA MEMBRANE PROFILING REVEALS A TARGETABLE CELL-SURFACE PHENOTYPE OF WNT-MEDULLOBLASTOMA

Abstract WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care surgical resection, radiotherapy, and chemotherapy. While curative, this treatment is associated with major, long-term, debilitating side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. The Gilbertson lab has previously shown that WNT-medulloblastomas are devoid of a Blood-Brain-Barrier (BBB). This disruption allows large molecules such as monoclonal antibodies (mAbs) to penetrate WNT-medulloblastomas. To discover WNT-medulloblastoma specific antigens for mAb targeting, we have first generated bulk and single-cell RNA sequencing profiles of the wild-type and WNT-medulloblastoma-prone mossy-fibre progenitor lineages in the lower rhombic lip, as well as human medulloblastoma patient derived orthotopic xenograft (PDOX) tumours. We next generated plasma membrane proteome profiles of hindbrain germinal zones and WNT-medulloblastomas. Plasma Membrane Profiling (PMP) deploys an isobaric chemical labelling approach -Tandem Mass Tags (TMT) - that allows multiplexing of up to sixteen variables per mass spectrometry experiment and ex vivo TMT-labelling of primary cells. Using this approach, we have mapped the cell surface proteome of our medulloblastoma models. Interrogation of these developmental and tumour data has unmasked WNT-medulloblastoma associated antigens that are specific for prenatal brain and WNT-medulloblastoma, but that are not expressed on critical postnatal tissues. Our workflow integrates multi-omic data sets to uncover a targetable landscape on the surface of WNT-medulloblastoma cells that we have cross-validated with a large cohort of patient RNA sequencing profiles and histological samples of both mouse and human tumours. To translate this dataset to real patient benefit, we are generating high affinity antibodies of these validated cell-surface proteins for assessment in our pre-clinical mouse models. Through our comparative analyses we have additionally identified non-WNT-specific targets and thus aim to expand this approach to identifying targets in other more agressive CNS tumours and brain metastases.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

MDB-06. PLASMA MEMBRANE PROFILING REVEALS A TARGETABLE CELL-SURFACE PHENOTYPE OF WNT-MEDULLOBLASTOMA

Abstract WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care surgical resection, radiotherapy, and chemotherapy. While curative, this treatment is associated with major, long-term, debilitating side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. The Gilbertson lab has previously shown that WNT-medulloblastomas are devoid of a Blood-Brain-Barrier (BBB). This disruption allows large molecules such as monoclonal antibodies (mAbs) to penetrate WNT-medulloblastomas. To discover WNT-medulloblastoma specific antigens for mAb targeting, we have first generated bulk and single-cell RNA sequencing profiles of the wild-type and WNT-medulloblastoma-prone mossy-fibre progenitor lineages in the lower rhombic lip, as well as human medulloblastoma patient derived orthotopic xenograft (PDOX) tumours. We next generated plasma membrane proteome profiles of hindbrain germinal zones and WNT-medulloblastomas. Plasma Membrane Profiling (PMP) deploys an isobaric chemical labelling approach -Tandem Mass Tags (TMT) - that allows multiplexing of up to sixteen variables per mass spectrometry experiment and ex vivo TMT-labelling of primary cells. Using this approach, we have mapped the cell surface proteome of our medulloblastoma models. Interrogation of these developmental and tumour data has unmasked WNT-medulloblastoma associated antigens that are specific for prenatal brain and WNT-medulloblastoma, but that are not expressed on critical postnatal tissues. Our workflow integrates multi-omic data sets to uncover a targetable landscape on the surface of WNT-medulloblastoma cells that we have cross-validated with a large cohort of patient RNA sequencing profiles and histological samples of both mouse and human tumours. To translate this dataset to real patient benefit, we are generating high affinity antibodies of these validated cell-surface proteins for assessment in our pre-clinical mouse models. mAbs directed towards essential receptors may have a therapeutic effect alone, however we will additionally modify mAbs to specifically deliver cytotoxic therapeutics - curing children whilst sparing healthy brain tissue.

A Peptidisc-Based Survey of the Plasma Membrane Proteome of a Mammalian Cell

Membrane proteins play critical roles at the cell surface and their misfunction is a hallmark of many human diseases. A precise evaluation of the plasma membrane proteome is therefore essential for cell biology and for discovering novel biomarkers and therapeutic targets. However, the low abundance of this proteome relative to soluble proteins makes it difficult to characterize, even with the most advanced proteomics technologies. Here, we apply the peptidisc membrane mimetic to purify the cell membrane proteome. Using the HeLa cell line as a reference, we capture 500 different integral membrane proteins, with half annotated to the plasma membrane. Notably, the peptidisc library is enriched with several ABC, SLC, GPCR, CD, and cell adhesion molecules that generally exist at low to very low copy numbers in the cell. We extend the method to compare two pancreatic cell lines, Panc-1 and hPSC. Here we observe a striking difference in the relative abundance of the cell surface cancer markers L1CAM, ANPEP, ITGB4, and CD70. We also identify two novel SLC transporters, SLC30A1 and SLC12A7, that are highly present in the Panc-1 cell only. The peptidisc library thus emerges as an effective way to survey and compare the membrane proteome of mammalian cells. Furthermore, since the method stabilizes membrane proteins in a water-soluble state, members of the library, here SLC12A7, can be specifically isolated.

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5 CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.

Membrane Proteomics to Understand Enhancement Effects of Millimeter-Wave Irradiation on Wheat Root under Flooding Stress.

Millimeter-wave irradiation of wheat seeds enhances the growth of roots under flooding stress, but its mechanism is not clearly understood. To understand the role of millimeter-wave irradiation on root-growth enhancement, membrane proteomics was performed. Membrane fractions purified from wheat roots were evaluated for purity. H+-ATPase and calnexin, which are protein markers for membrane-purification efficiency, were enriched in a membrane fraction. A principal-component analysis of the proteomic results indicated that the millimeter-wave irradiation of seeds affects membrane proteins in grown roots. Proteins identified using proteomic analysis were confirmed using immunoblot or polymerase chain reaction analyses. The abundance of cellulose synthetase, which is a plasma-membrane protein, decreased under flooding stress; however, it increased with millimeter-wave irradiation. On the other hand, the abundance of calnexin and V-ATPase, which are proteins in the endoplasmic reticulum and vacuolar, increased under flooding stress; however, it decreased with millimeter-wave irradiation. Furthermore, NADH dehydrogenase, which is found in mitochondria membranes, was upregulated due to flooding stress but downregulated following millimeter-wave irradiation even under flooding stress. The ATP content showed a similar trend toward change in NADH dehydrogenase expression. These results suggest that millimeter-wave irradiation improves the root growth of wheat via the transitions of proteins in the plasma membrane, endoplasmic reticulum, vacuolar, and mitochondria.

Identification of Membrane Protein Types Based Using Hypergraph Neural Network

Introduction: Membrane proteins play an important role in living organisms as one of the main components of biological membranes. The problem in membrane protein classification and prediction is an important topic of membrane proteomics research because the function of proteins can be quickly determined if membrane protein types can be discriminated. Methods: Most current methods to classify membrane proteins are labor-intensive and require a lot of resources. In this study, five methods, Average Block (AvBlock), Discrete Cosine Transform (DCT), Discrete Wavelet Transform (DWT), Histogram of Orientation Gradient (HOG), and Pseudo-PSSM (PsePSSM), were used to extract features in order to predict membrane proteins on a large scale. Then, we combined the five obtained feature matrices and constructed the corresponding hypergraph association matrix. Finally, the feature matrices and hypergraph association matrices were integrated to identify the types of membrane proteins using a hypergraph neural network model (HGNN). Results: The proposed method was tested on four membrane protein benchmark datasets to evaluate its performance. The results showed 92.8%, 88.6%, 88.2%, and 99.0% accuracy on each of the four datasets. Conclusion: Compared to traditional machine learning classifier methods, such as Random Forest (RF), Support Vector Machine (SVM), etc., HGNN prediction performance was found to be better.

A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

Abstract LB263: An in vivo unbiased screen identifies clathrin adaptor protein complex-2 as a novel tumor suppressor

Abstract Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly lethal. It is thus imperative to find new therapeutic approaches to treat this disease. While whole genome CRISPR/Cas9 screens have successfully identified new vulnerabilities in PDAC cell lines, it is becoming increasingly clear that cell culture do not faithfully recapitulate the complex nature of tumorigenesis in vivo, and hence may fail to identify the full spectrum of potential therapeutic targets to be explored in this devastating disease. As previous screens we performed identified phosphatidylinositol (Ptdlns) metabolism as important for pancreatic cell growth, I generated a custom sgRNA library targeting this entire form of metabolism. To identify vulnerabilities specific to tumor growth, I screened this library in five different human PDAC cell lines grown in culture versus as xenograft tumors. I found that sgRNAs targeting two genes encoding separate subunits of the Adaptor Protein complex-2 (AP2), a regulator of clathrin mediated endocytosis, were negatively enriched in all five PDAC cell lines when grown in culture. In agreement, AP2 subunit genes are considered common essential by DepMap. Paradoxically however, the same sgRNAs were positively enriched in three of these five cell lines when grown as xenograft tumors. This begs the question how the loss of a supposably essential complex behaves like a tumor suppressor and instead enhances tumor growth when disrupted. Here, I will present data that loss of AP2 results in a substantial restructuring the plasma membrane proteome, which in cultured cells results in the loss of iron transport, one of the main functions of this complex, and cell death. Conversely, this restructuring in vivo retains receptors and adhesion proteins on the cell surface, where transcriptome and proteomic analysis indicate enhances proliferative signaling. These data support AP2 promoting cell viability of cultured cells through active iron transport, but suppresses tumor growth in vivo by endocytosing the very receptors needed for tumor growth. Citation Format: Seth Parker Zimmerman, Christopher M. Counter. An in vivo unbiased screen identifies clathrin adaptor protein complex-2 as a novel tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB263.

ERV14 receptor impacts mycelial growth via its interactions with cell wall synthase and transporters in Aspergillus niger.

Efficient protein secretion is closely correlated with vesicle sorting and packaging, especially with cargo receptor-mediated selective transport for ER exit. Even though Aspergillus niger is considered an industrially natural host for protein production due to its exceptional secretion capacity, the trafficking mechanism in the early secretory pathway remains a black box for us to explore. Here, we identified and characterized all putative ER cargo receptors of the three families in A. niger. We successfully constructed overexpression and deletion strains of each receptor and compared the colony morphology and protein secretion status of each strain. Among them, the deletion of Erv14 severely inhibited mycelial growth and secretion of extracellular proteins such as glucoamylase. To gain a comprehensive understanding of the proteins associated with Erv14, we developed a high-throughput method by combining yeast two-hybrid (Y2H) with next-generation sequencing (NGS) technology. We found Erv14 specifically interacted with transporters. Following further validation of the quantitative membrane proteome, we determined that Erv14 was associated with the transport of proteins involved in processes such as cell wall synthesis, lipid metabolism, and organic substrate metabolism.

Multi-omic characterization of the maize GPI synthesis mutant gwt1 with defects in kernel development

BackgroundGlycosylphosphatidylinositol (GPI) and GPI-anchored proteins (GAPs) are important for cell wall formation and reproductive development in Arabidopsis. However, monocot counterparts that function in kernel endosperm development have yet to be discovered. Here, we performed a multi-omic analysis to explore the function of GPI related genes on kernel development in maize.ResultsIn maize, 48 counterparts of human GPI synthesis and lipid remodeling genes were identified, in which null mutation of the glucosaminyl-phosphatidylinositol O-acyltransferase1 gene, ZmGWT1, caused a kernel mutant (named gwt1) with defects in the basal endosperm transport layer (BETL). We performed plasma membrane (PM) proteomics to characterize the potential GAPs involved in kernel development. In total, 4,981 proteins were successfully identified in 10-DAP gwt1 kernels of mutant and wild-type (WT), including 1,638 membrane-anchored proteins with different posttranslational modifications. Forty-seven of the 256 predicted GAPs were differentially accumulated between gwt1 and WT. Two predicted BETL-specific GAPs (Zm00001d018837 and Zm00001d049834), which kept similar abundance at general proteome but with significantly decreased abundance at membrane proteome in gwt1 were highlighted.ConclusionsOur results show the importance of GPI and GAPs for endosperm development and provide candidate genes for further investigation of the regulatory network in which ZmGWT1 participates.

Abstract 2646: Screening the membrane proteome to determine antibody specificity and de-risk CAR-T cell development

Abstract Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity. We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in live cells. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. We will discuss the importance of early-stage specificity testing to expose possible toxicities and present case studies for antibody and cell therapy profiling. MPA data have been used in numerous successful regulatory applications and may be used to replace or complement other cross-reactivity studies. In particular, MPA profiling has been used in IND applications for CAR-T and cell-therapies where conventional immunohistochemical binding assays did not suffice. Citation Format: Joseph Rucker, Rachel H. Fong, Tabb Sullivan, Carmen Navia, Brad Screnci, Rona Wilf, Benjamin J. Doranz. Screening the membrane proteome to determine antibody specificity and de-risk CAR-T cell development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2646.