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The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential. In polarized epithelial cells, GPI-APs are enriched at the apical surface, where they form small cholesterol-independent homoclusters and larger heteroclusters accommodating multiple GPI-AP species, all confined within areas of approximately 65–70 nm in diameter. Notably, GPI-AP homoclustering occurs in the Golgi apparatus through a cholesterol- and calcium-dependent mechanism that drives their apical sorting. Despite the critical role of Golgi GPI-AP clustering in their cell surface organization and the importance of cholesterol in heterocluster formation, the regulatory mechanisms governing GPI-AP surface organization, particularly in the context of epithelial polarity, remain elusive. Given that the actin cytoskeleton undergoes substantial remodeling during polarity establishment, this study explores whether the actin cytoskeleton regulates the spatiotemporal apical organization of GPI-APs in MDCK cells. Utilizing various imaging techniques (number and brightness, FRET/FLIM, and dSTORM coupled to pair correlation analysis), we demonstrate that the apical organization of GPI-APs, at different scales, does not rely on the actin cytoskeleton, unlike in fibroblastic cells. Interestingly, calcium chelation disrupts the organization of GPI-APs at the apical surface by impairing Golgi GPI-AP clustering, emphasizing the existence of an interplay among Golgi clustering, apical sorting, and surface organization in epithelial cells. In summary, our findings unveil distinct mechanisms regulating the organization of GPI-APs in cell types of different origins, plausibly allowing them to adapt to different external signals and different cellular environments in order to achieve specialized functions.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed. [ABSTRACT FROM AUTHOR]

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the viability of upper and lower motor neurons. Current options for treatment are limited, necessitating deeper understanding of the mechanisms underlying ALS pathogenesis. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that acts on the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. GDE2 is required for the survival of spinal motor neurons but whether GDE2 neuroprotective activity is disrupted in ALS is not known. We utilized a combination of mouse models and patient post-mortem samples to evaluate GDE2 functionality in ALS. Haplogenetic reduction of GDE2 exacerbated motor neuron degeneration and loss in SOD1 G93A mice but not in control SOD1 WT transgenic animals, indicating that GDE2 neuroprotective function is diminished in the context of SOD1 G93A. In tissue samples from patients with ALS, total levels of GDE2 protein were equivalent to healthy controls; however, membrane levels of GDE2 were substantially reduced. Indeed, GDE2 was found to aberrantly accumulate in intracellular compartments of ALS motor cortex, consistent with a disruption of GDE2 function at the cell surface. Supporting the impairment of GDE2 activity in ALS, tandem-mass-tag mass spectrometry revealed a pronounced reduction of GPI-anchored proteins released into the CSF of patients with ALS compared with control patients. Taken together, this study provides cellular and biochemical evidence that GDE2 distribution and activity is disrupted in ALS, supporting the notion that the failure of GDE2-dependent neuroprotective pathways contributes to neurodegeneration and motor neuron loss in disease. These observations highlight the dysregulation of GPI-anchored protein pathways as candidate mediators of disease onset and progression and accordingly, provide new insight into the mechanisms underlying ALS pathogenesis.

Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single β-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal 'pre-orientation' of the LU domain for the receptor interaction. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the viability of upper and lower motor neurons. Current options for treatment are limited, necessitating deeper understanding of the mechanisms underlying ALS pathogenesis. Glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that acts on the cell surface to cleave the glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane. GDE2 is required for the survival of spinal motor neurons but whether GDE2 neuroprotective activity is disrupted in ALS is not known. We utilized a combination of mouse models and patient post-mortem samples to evaluate GDE2 functionality in ALS. Haplogenetic reduction of GDE2 exacerbated motor neuron degeneration and loss in SOD1G93A mice but not in control SOD1WT transgenic animals, indicating that GDE2 neuroprotective function is diminished in the context of SOD1G93A. In tissue samples from patients with ALS, total levels of GDE2 protein were equivalent to healthy controls; however, membrane levels of GDE2 were substantially reduced. Indeed, GDE2 was found to aberrantly accumulate in intracellular compartments of ALS motor cortex, consistent with a disruption of GDE2 function at the cell surface. Supporting the impairment of GDE2 activity in ALS, tandem-mass-tag mass spectrometry revealed a pronounced reduction of GPI-anchored proteins released into the CSF of patients with ALS compared with control patients. Taken together, this study provides cellular and biochemical evidence that GDE2 distribution and activity is disrupted in ALS, supporting the notion that the failure of GDE2-dependent neuroprotective pathways contributes to neurodegeneration and motor neuron loss in disease. These observations highlight the dysregulation of GPI-anchored protein pathways as candidate mediators of disease onset and progression and accordingly, provide new insight into the mechanisms underlying ALS pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract Background Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial. Results We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains. Conclusion Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins’ mechanism of action in a super pest.

The protozoa Leishmania donovani causes visceral leishmaniasis (kala-azar), the third most common vector-borne disease. The visceral organs, particularly the spleen, liver, and bone marrow, are affected by the disease. The lack of effective treatment regimens makes curing and eradicating the disease difficult. The availability of complete L. donovani genome/proteome data allows for the development of specific and efficient vaccine candidates using the reverse vaccinology method, while utilizing the unique sequential and structural features of potential antigenic proteins to induce protective T cell and B cell responses. Such shortlisted candidates may then be tested quickly for their efficacy in the laboratory and later in clinical settings. These antigens will also be useful for designing antigen-based next-generation sero-diagnostic assays. L. donovani's cell surface-associated proteins and secretory proteins are among the first interacting entities to be exposed to the host immune machinery. As a result, potential antigenic epitope peptides derived from these proteins could serve as competent vaccine components. We used a stepwise filtering-based in silico approach to identify the entire surface-associated and secretory proteome of L. donovani, which may provide rationally selected most exposed antigenic proteins. Our study identified 12 glycosylphosphatidylinositol-anchored proteins, 45 transmembrane helix-containing proteins, and 73 secretory proteins as potent antigens unique to L. donovani. In addition, we used immunoinformatics to identify B and T cell epitopes in them. Out of the shortlisted surface-associated and secretory proteome, 66 protein targets were found to have the most potential overlapping B cell and T cell epitopes (linear and conformational; MHC class I and MHC class II). [ABSTRACT FROM AUTHOR]

Aspergillosis, candidiasis, and cryptococcosis are the most common cause of mycoses-related disease and death among immune-compromised patients. Adhesins are cell-surface exposed proteins or glycoproteins of pathogens that bind to the extracellular matrix (ECM) constituents or mucosal epithelial surfaces of the host cells. The forces of interaction between fungal adhesins and host tissues are accompanied by ligand binding, hydrophobic interactions and protein-protein aggregation. Adherence is the primary and critical step involved in the pathogenesis; however, there is limited information on fungal adhesins compared to that on the bacterial adhesins. Except a few studies based on screening of proteome for adhesin identification, majority are based on characterization of individual adhesins. Recently, based on their characteristic signatures, many putative novel fungal adhesins have been predicted using bioinformatics algorithms. Some of these novel adhesin candidates have been validated by in-vitro studies; though, most of them are yet to be characterised experimentally. Morphotype specific adhesin expression as well as tissue tropism are the crucial determinants for a successful adhesion process. This review presents a comprehensive overview of various studies on fungal adhesins and discusses the targetability of the adhesins and adherence phenomenon, for combating the fungal infection in a preventive or therapeutic mode.

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed.

Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1 / PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus. [ABSTRACT FROM AUTHOR]

Background: Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial. Results: We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains. Conclusion: Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins’ mechanism of action in a super pest. [ABSTRACT FROM AUTHOR]

Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1/PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus.

Complexes of p24 proteins act as cargo receptors for the transport of COPII vesicles from the endoplasmic reticulum (ER). The major cargos of p24 complexes are hydrophilic proteins tethered to the ER membrane via a covalently attached glycosylphosphatidylinositol (GPI) or fatty acid. Each p24 complex is known to contain members from all four p24 subfamilies (p24α, p24β, p24γ and p24δ). However, it remains unclear how the cargo specificities of p24 complexes are influenced by member stoichiometry. Here, we report the subunit compositions of mammalian p24 complexes involved in the transport of GPI-anchored proteins and Wnt1. We show that at least one p24α is required for the formation of p24 complexes and that a p24 complex consisting of p24α2, p24β1, p24γ2 and p24δ1 is required for the efficient transport of GPI-anchored proteins. On the other hand, a p24 complex containing p24α2, p24α3, p24β1, p24γ and p24δ1 is involved in the transport of Wnt1. Further, interactions between p24α2 and p24α3 are critical for Wnt1 transport. Thus, p24α and p24γ subfamily members are important for cargo selectivity. Lastly, our data fit with an octamer, rather than a tetramer, model of p24 complexes, where each complex consists of two proteins from each p24 subfamily. [ABSTRACT FROM AUTHOR]

Aspergillosis, candidiasis, and cryptococcosis are the most common cause of mycoses-related disease and death among immune-compromised patients. Adhesins are cell-surface exposed proteins or glycoproteins of pathogens that bind to the extracellular matrix (ECM) constituents or mucosal epithelial surfaces of the host cells. The forces of interaction between fungal adhesins and host tissues are accompanied by ligand binding, hydrophobic interactions and protein-protein aggregation. Adherence is the primary and critical step involved in the pathogenesis; however, there is limited information on fungal adhesins compared to that on the bacterial adhesins. Except a few studies based on screening of proteome for adhesin identification, majority are based on characterization of individual adhesins. Recently, based on their characteristic signatures, many putative novel fungal adhesins have been predicted using bioinformatics algorithms. Some of these novel adhesin candidates have been validated by in-vitro studies; though, most of them are yet to be characterised experimentally. Morphotype specific adhesin expression as well as tissue tropism are the crucial determinants for a successful adhesion process. This review presents a comprehensive overview of various studies on fungal adhesins and discusses the targetability of the adhesins and adherence phenomenon, for combating the fungal infection in a preventive or therapeutic mode. [ABSTRACT FROM AUTHOR]

Early infantile epileptic encephalopathy 38 (EIEE38, MIM #617020) is caused by biallelic variants in ARV1, encoding a transmembrane protein of the endoplasmic reticulum with a pivotal role in glycosylphosphatidylinositol (GPI) biosynthesis. We ascertained seven new patients from six unrelated families harboring biallelic variants in ARV1, including five novel variants. Affected individuals showed psychomotor delay, hypotonia, early onset refractory seizures followed by regression and specific neuroimaging features. Flow cytometric analysis on patient fibroblasts showed a decrease in GPI‐anchored proteins on the cell surface, supporting a lower residual activity of the mutant ARV1 as compared to the wildtype. A rescue assay through the transduction of lentivirus expressing wild type ARV1 cDNA effectively rescued these alterations. This study expands the clinical and molecular spectrum of the ARV1‐related encephalopathy, confirming the essential role of ARV1 in GPI biosynthesis and brain function. [ABSTRACT FROM AUTHOR]

Lipid and protein diversity provides structural and functional identity to the membrane compartments that define the eukaryotic cell. This compositional heterogeneity is maintained by the secretory pathway, which feeds newly synthesized proteins and lipids to the endomembrane systems. The precise sorting of lipids and proteins through the pathway guarantees the achievement of their correct delivery. Although proteins have been shown to be key for sorting mechanisms, whether and how lipids contribute to this process is still an open discussion. Our laboratory, in collaboration with other groups, has recently addressed the long-postulated role of membrane lipids in protein sorting in the secretory pathway, by investigating in yeast how a special class of lipid-linked cell surface proteins are differentially exported from the endoplasmic reticulum. Here we comment on this interdisciplinary study that highlights the role of lipid diversity and the importance of protein-lipid interactions in sorting processes at the cell membrane.

Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single β-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal ‘pre-orientation’ of the LU domain for the receptor interaction.

The spatiotemporal compartmentalization of membrane-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell surface regulates their biological activities. These GPI-APs occupy distinct cellular functions such as enzymes, receptors, and adhesion molecules, and they are implicated in several vital cellular processes. Thus, unraveling the mechanisms and regulators of their membrane organization is essential. In polarized epithelial cells, GPI-APs are enriched at the apical surface, where they form small cholesterol-independent homoclusters and larger heteroclusters accommodating multiple GPI-AP species, all confined within areas of approximately 65-70 nm in diameter. Notably, GPI-AP homoclustering occurs in the Golgi apparatus through a cholesterol- and calcium-dependent mechanism that drives their apical sorting. Despite the critical role of Golgi GPI-AP clustering in their cell surface organization and the importance of cholesterol in heterocluster formation, the regulatory mechanisms governing GPI-AP surface organization, particularly in the context of epithelial polarity, remain elusive. Given that the actin cytoskeleton undergoes substantial remodeling during polarity establishment, this study explores whether the actin cytoskeleton regulates the spatiotemporal apical organization of GPI-APs in MDCK cells. Utilizing various imaging techniques (number and brightness, FRET/FLIM, and dSTORM coupled to pair correlation analysis), we demonstrate that the apical organization of GPI-APs, at different scales, does not rely on the actin cytoskeleton, unlike in fibroblastic cells. Interestingly, calcium chelation disrupts the organization of GPI-APs at the apical surface by impairing Golgi GPI-AP clustering, emphasizing the existence of an interplay among Golgi clustering, apical sorting, and surface organization in epithelial cells. In summary, our findings unveil distinct mechanisms regulating the organization of GPI-APs in cell types of different origins, plausibly allowing them to adapt to different external signals and different cellular environments in order to achieve specialized functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lebreton, Paladino, Lelek, Tramier, Zimmer and Zurzolo.)

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi. [ABSTRACT FROM AUTHOR]

Abstract Background Gastric cancer is one of the deadliest malignant tumours, with a high incidence in China, and is regulated by aberrantly overexpressed oncogenes. However, existing therapies are insufficient to meet patients’ needs; thus, the identification of additional therapeutic targets and exploration of the underlying mechanism are urgently needed. GPAA1 is the subunit of the GPI transamidase that transfers the GPI anchor to proteins within the ER. The functional impacts of increased expression levels of GPAA1 in human cancers are not well understood. Methods Data mining was performed to determine the pattern of GPAA1 expression and the reason for its overexpression in tumour and adjacent normal tissues. In vitro and in vivo experiments evaluating proliferation and metastasis were performed using cells with stable deletion or overexpression of GPAA1. A tissue microarray established by the Ren Ji Hospital was utilized to analyse the expression profile of GPAA1 and its correlation with prognosis. Western blotting, an in situ proximity ligation assay, and co-immunoprecipitation (co-IP) were performed to reveal the mechanism of GPAA1 in gastric cancer. Results GPAA1 was a markedly upregulated oncogene in gastric cancer due to chromosomal amplification. GPAA1 overexpression was confirmed in specimens from the Ren Ji cohort and was associated with ERBB2 expression, predicting unsatisfactory patient outcomes. Aberrantly upregulated GPAA1 dramatically contributed to cancer growth and metastasis in in vitro and in vivo studies. Mechanistically, GPAA1 enhanced the levels of metastasis-associated GPI-anchored proteins to increase tumour metastasis and intensified lipid raft formation, which consequently promoted the interaction between EGFR and ERBB2 as well as downstream pro-proliferative signalling. Conclusions GPAA1 facilitates the expression of cancer-related GPI-anchored proteins and supplies a more robust platform—the lipid raft—to promote EGFR-ERBB2 dimerization, which further contributes to tumour growth and metastasis and to cancer progression. GPAA1 could be a promising diagnostic biomarker and therapeutic target for gastric cancer.

Significance: Selenoproteins incorporate the essential nutrient selenium into their polypeptide chain. Seven members of this family reside in the endoplasmic reticulum (ER), the exact function of most of which is poorly understood. Especially, how ER-resident selenoproteins control the ER redox and ionic environment is largely unknown. Since alteration of ER function is observed in many diseases, the elucidation of the role of selenoproteins could enhance our understanding of the mechanisms involved in ER homeostasis. Recent Advances: Among selenoproteins, selenoprotein T (SELENOT) is remarkable as the most evolutionarily conserved and the only ER-resident selenoprotein whose gene knockout in mouse is lethal. Recent data indicate that SELENOT contributes to ER homeostasis: reduced expression of SELENOT in transgenic cell and animal models promotes accumulation of reactive oxygen and nitrogen species, depletion of calcium stores, activation of the unfolded protein response and impaired hormone secretion. Critical Issues: SELENOT is anchored to the ER membrane and associated with the oligosaccharyltransferase complex, suggesting that it regulates the early steps of N-glycosylation. Furthermore, it exerts a selenosulfide oxidoreductase activity carried by its thioredoxin-like domain. However, the physiological role of the redox activity of SELENOT is not fully understood. Likewise, the nature of its redox partners needs to be further characterized. Future Directions: Given the impact of ER stress in pathologies such as neurodegenerative, cardiovascular, metabolic and immune diseases, understanding the role of SELENOT and developing derived therapeutic tools such as selenopeptides to improve ER proteostasis and prevent ER stress could contribute to a better management of these diseases. [ABSTRACT FROM AUTHOR]

Members of the CAP/SCP/TAPS superfamily have been implicated in many different physiological processes, including pathogen defense, sperm maturation and fertilization. The mode of action of this class of proteins, however, remains poorly understood. The genome of Saccharomyces cerevisiae encodes three CAP superfamily members, Pry1-3. We have previously shown that Pry1 function is required for the secretion of sterols and fatty acids. Here, we analyze the function of Pry3, a GPI-anchored cell wall protein. Overexpression of Pry3 results in strong reduction of mating efficiency, providing for a cell-based readout for CAP protein function. Mating inhibition is a conserved function of the CAP domain and depends on highly conserved surface exposed residues that form part of a putative catalytic metal-ion binding site. Pry3 displays polarized cell surface localization adjacent to bud scars, but is absent from mating projections. When overexpressed, however, the protein leaks onto mating projections, suggesting that mating inhibition is due to mislocalization of the protein. Trapping of the CAP domain within the cell wall through a GPI-anchored nanobody results in a dose-dependent inhibition of mating, suggesting that a membrane proximal CAP domain inhibits a key step in the mating reaction, which is possibly related to the function of CAP domain proteins in mammalian fertilization. This article has an associated First Person interview with the first author of the paper.

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of β-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii, which had naturally more β-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and β-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells.

The cellular prion protein (PrPC) is renowned for its infectious conformational isoform PrPSc, capable of templating subsequent conversions of healthy PrPCs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein’s physiological role is also elusive. PrPC and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein–protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.

Exosomes are 50–100 nm membranous vesicles actively released by cells which can be indicative of a diseased cell status. They contain various kinds of molecule – proteins, mRNA, miRNA, lipids – that are actively being studied as potential biomarkers. Hereafter I put forward several arguments in favor of the potential use of glycosylphosphatidylinositol-anchored proteins (GPI-APs) as biomarkers especially of cancerous diseases. I will briefly update readers on the exosome field and review various features of GPI-APs, before further discussing the advantages of this class of proteins as potential exosomal biomarkers. I will finish with a few examples of exosomal GPI-APs that have already been demonstrated to be good prognostic markers, as well as innovative approaches developed to quantify these exosomal biomarkers. Unlabelled Image • Exosomes are extracellular vesicles (80-100 nm) derived from an endosomal compartment. • GPI-anchored proteins are overexpressed in various cancers and can often be used as biomarkers. • GPI-anchored proteins are efficiently sorted in exosomes and are present in meaningful amounts. • GPI-anchored proteins are exposed on the surface of exosomes and thus easily quantified. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositols (GPIs) anchored proteins are commonly localized onto lipid rafts. These extracellular proteins participate in a variety of cellular functions, including as receptors for viruses and toxins. Intracellular trafficking of World Health Organization recognized mosquito-larvicidal BinAB toxin is mediated via GPI-anchored Cqm1 receptor protein in Culex mosquitoes. We confirmed conformational change in Cqm1 dimer on interaction with BinA/BinB proteins by dynamic light scattering, modelling of hydrodynamic parameters using the atomic structures, and synchrotron Small Angle solution X-ray scattering (SAXS). A reliable model of the receptor-BinB complex was also constructed from joint SAXS/SANS refinement. We confirmed electrostatic interactions of the Cqm1 ectodomain with lipid rafts reconstituted in model membranes and report receptor-dependent impairment of model liposomes by BinA/B proteins. Liposomal disruption was toxin concentration-dependent as monitored by the release of encapsulated carboxyfluorescein dye. Interestingly, BinA alone, without BinB, showed efficient efflux of the fluorescent dye in agreement with the reported high larvicidal activity of BinA variants. The study provides insight into BinA/B toxin internalization mechanism in the membrane model that is toxin internalization is mediated via receptor-dependent pore formation mechanism. It also suggests a tangible and environmentally safe strategy for control of mosquito population. [ABSTRACT FROM AUTHOR]

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host–pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI‐anchored proteins (GPI‐APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well‐characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N‐terminal signal peptide and a single C‐terminal transmembrane region containing a GPI anchor signal. Twenty‐four GPI‐anchored peptides were identified, of which nine are likely S. aucheniae‐specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI‐anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra‐ and inter‐specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI‐APs as drug targets and/or as vaccine or diagnostic antigens. [ABSTRACT FROM AUTHOR]

Our understanding of the plasma membrane structure has undergone a major change since the proposal of the fluid mosaic model of Singer and Nicholson in the 1970s. In this model, the membrane, composed of over thousand lipid and protein species, is organized as a well‐equilibrated two‐dimensional fluid. Here, the distribution of lipids is largely expected to reflect a multicomponent system, and proteins are expected to be surrounded by an annulus of specialized lipid species. With the recognition that a multicomponent lipid membrane is capable of phase segregation, the membrane is expected to appear as patchwork quilt pattern of membrane domains. However, the constituents of a living membrane are far from being well equilibrated. The living cell membrane actively maintains a trans‐bilayer asymmetry of composition, and its constituents are subject to a number of dynamic processes due to synthesis, lipid transfer as well as membrane traffic and turnover. Moreover, membrane constituents engage with the dynamic cytoskeleton of a living cell, and are both passively as well as actively manipulated by this engagement. The extracellular matrix and associated elements also interact with membrane proteins contributing to another layer of interaction. At the nano‐ and mesoscale, the organization of lipids and proteins emerge from these encounters, as well as from protein–protein, protein–lipid, and lipid–lipid interactions in the membrane. New methods to study the organization of membrane components at these scales have also been developed, and provide an opportunity to synthesize a new picture of the living cell surface as an active membrane composite. [ABSTRACT FROM AUTHOR]

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of β-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii , which had naturally more β-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and β-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells. [ABSTRACT FROM AUTHOR]

Sexually reproducing organisms generate male and female haploid gametes, which meet and fuse at fertilization to produce a diploid zygote. The evolutionary process of speciation is achieved and maintained by ensuring that gametes undergo productive fusion only within a species. In animals, hybrids from cross-species fertilization events may develop normally, but are usually sterile (Fitzpatrick, 2004). Metazoan sperm and eggs have several features to ensure that the gametes, which have evolved independently and also in conflict with each other, are competent to undergo fertilization (Firman, 2018). Fertilization is a specific process that is ideally supposed to result in randomized fusion of compatible egg and sperm. Here, I will discuss key processes driven by maternal factors in the egg that dictate earliest stages of gamete recognition, gamete choice and fusion in metazoans. [ABSTRACT FROM AUTHOR]

Pollen tube integrity is required for achieving double fertilization in angiosperms. The rapid alkalinization factor4/19-ANXUR1/2-Buddha's paper seal 1/2 (RALF4/19-ANX1/2-BUPS1/2)-complex-mediated signaling pathway is critical to maintain pollen tube integrity, but the underlying mechanisms regulating the polar localization and distribution of these complex members at the pollen tube tip remain unclear. Here, we find that COBRA-like protein 11 (COBL11) loss-of-function mutants display a low pollen germination ratio, premature pollen tube burst, and seed abortion in Arabidopsis. COBL11 could interact with RALF4/19, ANX1/2, and BUPS1/2, and COBL11 functional deficiency could result in the disrupted distribution of RALF4 and ANX1, altered cell wall composition, and decreased levels of reactive oxygen species in pollen tubes. In conclusion, COBL11 is a regulator of pollen tube integrity during polar growth, which is conducted by a direct interaction that ensures the correct localization and polar distribution of RALF4 and ANX1 at the pollen tube tip. [Display omitted] • COBL11, a member of COBRA subfamily, contributes to pollen tube integrity • COBL11 functional deficiency causes mild male sterility • COBL11 interacts with RALF-ANX-BUPS complex members • Loss of COBL11 function affects ROS levels and cell wall composition COBRA and COBL (COBRA-like) are a phylogenetic family encoding plant-specific GPI-APs in Arabidopsis. Here, Li et al. report that COBL11 regulates pollen tube integrity by interacting with RALF4/19, ANX1/2, and BUPS1/2 to maintain their appropriate locations and maintaining their homeostasis and polar distribution. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Membrane proteins can be classified into two main categories—integral and peripheral membrane proteins—depending on the nature of their membrane interaction. Peripheral membrane proteins are highly unique amphipathic proteins that interact with the membrane indirectly, using electrostatic or hydrophobic interactions, or directly, using hydrophobic tails or GPI-anchors. The nature of this interaction not only influences the location of the protein in the cell, but also the function. In addition to their unique relationship with the cell membrane, peripheral membrane proteins often play a key role in the development of human diseases such as African sleeping sickness, cancer, and atherosclerosis. This review will discuss the membrane interaction and role of periplasmic nitrate reductase, CymA, cytochrome c, alkaline phosphatase, ecto-5’-nucleotidase, acetylcholinesterase, alternative oxidase, type-II NADH dehydrogenase, and dihydroorotate dehydrogenase in certain diseases. The study of these proteins will give new insights into their function and structure, and may ultimately lead to ground-breaking advances in the treatment of severe diseases.

Abstract Background Glycophosphatidylinositol‐anchored proteins (GPI‐APs) mediate several physiological processes such as embryogenesis and neurogenesis. Germline variants in genes involved in their synthesis can disrupt normal development and result in a variety of clinical phenotypes. With the advent of new sequencing technologies, more cases are identified, leading to a rapidly growing number of reported genetic variants. With this number expected to rise with increased accessibility to molecular tests, an accurate and up‐to‐date database is needed to keep track of the information and help interpret results. Methods We therefore developed an online resource (www.gpibiosynthesis.org) which compiles all published pathogenic variants in GPI biosynthesis genes which are deposited in the LOVD database. It contains 276 individuals and 192 unique public variants; 92% of which are predicted as damaging by bioinformatics tools. Results A significant proportion of recorded variants was substitution variants (81%) and resulted mainly in missense and frameshift alterations. Interestingly, five patients (2%) had deleterious mutations in untranslated regions. CADD score analysis placed 97% of variants in the top 1% of deleterious variants in the human genome. In genome aggregation database, the gene with the highest frequency of reported pathogenic variants is PIGL, with a carrier rate of 1/937. Conclusion We thus present the GPI biosynthesis database and review the molecular genetics of published variants in GPI‐anchor biosynthesis genes.

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are lipid APs attached to the extracellular leaflet of the plasma membrane (PM) via a glycolipid anchor. GPI‐APs are commonly associated with cholesterol‐ and sphingolipid‐enriched membrane microdomains. These microdomains help regulating various biological activities, by segregating different proteins and lipids in (nanoscale) membrane compartments. In fibroblasts, GPI‐APs form actin‐ and cholesterol‐dependent nanoclusters directly at the PM. In contrast, in polarized epithelial cells GPI‐APs cluster in the Golgi apparatus, the major protein‐sorting hub for the secretory pathway. Golgi clustering is required for the selective sorting of GPI‐APs to the apical PM domain, but also regulates their organization and biological activities at the cell surface. In this review, we discuss recent advances in our understanding of the mechanism of GPI‐AP sorting to the apical membrane. We focus on the roles of the protein moiety and lipids in the regulation of the clustering of GPI‐APs in the Golgi apparatus. [ABSTRACT FROM AUTHOR]

It is estimated that 0.5% of all mammalian proteins have a glycosylphosphatidylinositol (GPI)‐anchor. GPI‐anchored proteins (GPI‐APs) play key roles, particularly in embryogenesis, neurogenesis, immune response and signal transduction. Due to their involvement in many pathways and developmental events, defects in the genes involved in their synthesis and processing can result in a variety of genetic disorders for which affected individuals display a wide spectrum of features. We compiled the clinical characteristics of 202 individuals with mutations in the GPI biosynthesis and processing pathway through a review of the literature. This review has allowed us to compare the characteristics and the severity of the phenotypes associated with different genes as well as highlight features that are prominent for each. Certain combinations, such as seizures with aplastic/hypoplastic nails or abnormal alkaline phosphatase levels suggest an inherited GPI deficiency, and our review of all clinical findings may orient the management of inherited GPI deficiencies. [ABSTRACT FROM AUTHOR]

Abstract An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L o phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different – the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts. Graphical abstract Unlabelled Image Highlights • SMA disintegrates T cell membrane into SMALPs. • Most membrane proteins are present in small SMALPs. • Some membrane raft-associated proteins are present in large SMALPs. • The large SMALPs have specific lipid composition corresponding to membrane rafts. • T cell membrane proteins present in SMALPs can be readily immunoisolated. [ABSTRACT FROM AUTHOR]

The COBRA-like (COBL) gene family has been associated with the regulation of cell wall expansion and cellulose deposition. COBL mutants result in reduced levels and disorganized deposition of cellulose causing defects in the cell wall and inhibiting plant development. In this study, we report the identification of 24 COBL genes (GmCOBL) in the soybean genome. Phylogenetic analysis revealed that the COBL proteins are divided into two groups, which differ by about 170 amino acids in the N-terminal region. The GmCOBL genes were heterogeneously distributed in 14 of the 20 soybean chromosomes. This study showed that segmental duplication has contributed significantly to the expansion of the COBL family in soybean during all Glycine-specific whole-genome duplication events. The expression profile revealed that the expression of the paralogous genes is highly variable between organs and tissues of the plant. Only 20% of the paralogous gene pairs showed similar expression patterns. The high expression levels of some GmCOBLs suggest they are likely essential for regulating cell expansion during the whole soybean life cycle. Our comprehensive overview of the COBL gene family in soybean provides useful information for further understanding the evolution and diversification of COBL genes in soybean.

PIGT encodes a subunit of the glycosylphosphatidylinositol transamidase complex, which catalyzes the attachment of proteins to GPI-anchors. A homozygous PIGT variant c.550G>A (p. E184K) in a Chinese boy with multiple malformations, hypotonia, seizure and profound development delay was identified by panel sequencing. Pathogenicity of the variant was confirmed by flow cytometry. The expression of CD16 and CD24 of this proband reduced to 16.92 and 22.16% compare with normal control respectively while which of his parents and sister were normal. This mutation raised the mRNA level on the peripheral blood mono nuclear cells of this patient. This study expanded the variant spectrum of MCAHS3, and CD16 could be an effective marker to evaluate the pathogenicity of PIGT mutation.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gonçales, Portis, dos Reis, Basso Júnior, Martinez, Zhu, Pereira, Soares and Coelho.)

Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrPC, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer. [ABSTRACT FROM AUTHOR]

PIGT encodes a subunit of the glycosylphosphatidylinositol transamidase complex, which catalyzes the attachment of proteins to GPI-anchors. A homozygous PIGT variant c.550G>A (p. E184K) in a Chinese boy with multiple malformations, hypotonia, seizure and profound development delay was identified by panel sequencing. Pathogenicity of the variant was confirmed by flow cytometry. The expression of CD16 and CD24 of this proband reduced to 16.92 and 22.16% compare with normal control respectively while which of his parents and sister were normal. This mutation raised the mRNA level on the peripheral blood mono nuclear cells of this patient. This study expanded the variant spectrum of MCAHS3, and CD16 could be an effective marker to evaluate the pathogenicity of PIGT mutation. [ABSTRACT FROM AUTHOR]

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol‐anchored proteins (GPI‐APs) and soluble secretory proteins in Madin‐Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI‐APs in biosynthetic pathway but also for their apical recycling and basal‐to‐apical transcytosis routes. The apical distribution of the t‐SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical‐destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells. [ABSTRACT FROM AUTHOR]

Abstract: As biochemical and functional studies of membrane protein remain a challenge, there is growing interest in the application of nanotechnology to solve the difficulties of developing membrane protein therapeutics. Exosome, composed of lipid bilayer enclosed nanosized extracellular vesicles, is a successful platform for providing a native membrane composition. This study reports an enzymatic exosome, which harbors native PH20 hyaluronidase (Exo‐PH20), which is able to penetrate deeply into tumor foci via hyaluronan degradation, allowing tumor growth inhibition and increased T cell infiltration into the tumor. This exosome‐based strategy is developed to overcome the immunosuppressive and anticancer therapy‐resistant tumor microenvironment, which is characterized by an overly accumulated extracellular matrix. Notably, this engineered exosome with the native glycosylphosphatidylinositol‐anchored form of hyaluronidase has a higher enzymatic activity than a truncated form of the recombinant protein. In addition, the exosome‐mediated codelivery of PH20 hyaluronidase and a chemotherapeutic (doxorubicin) efficiently inhibits tumor growth. This exosome is designed to degrade hyaluronan, thereby augmenting nanoparticle penetration and drug diffusion. The results thus show that this is a promising exosome‐based platform that harbors not only a membrane‐associated enzyme with high activity but also therapeutic payloads. [ABSTRACT FROM AUTHOR]

Two key proteins for cellular communication between astrocytes and neurons are αvβ3 integrin and the receptor Thy-1. Binding of these molecules in the same ( cis ) or on adjacent ( trans ) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvβ3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvβ3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 ( trans ) to the neuronal actin cytoskeleton. Importantly, the β3 integrin in neurons ( cis ) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvβ3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication. [ABSTRACT FROM AUTHOR]