SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, i.e., Lck, Fyn, Lyn, Hck, Fgr, Blk, Src, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that nucleates an autoinhibitory complex. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (Pep/LYP), which dephosphorylate the SFK activation loop to promote autoinhibition. High local concentrations of Csk are required to promote its negative-regulatory function, and Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains. Purified Csk also homodimerizes in solution through an interface that overlaps the phosphatase binding site. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Csk W47A, an SH3-domain mutant commonly used to block PTPN22 binding, also impairs homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with both Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

Cholesterol-Rich Lipid Rafts as Platforms for SARS-CoV-2 Entry

Since its appearance, the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), the causal agent of Coronavirus Disease 2019 (COVID-19), represents a global problem for human health that involves the host lipid homeostasis. Regarding, lipid rafts are functional membrane microdomains with highly and tightly packed lipid molecules. These regions enriched in sphingolipids and cholesterol recruit and concentrate several receptors and molecules involved in pathogen recognition and cellular signaling. Cholesterol-rich lipid rafts have multiple functions for viral replication; however, their role in SARS-CoV-2 infection remains unclear. In this review, we discussed the novel evidence on the cholesterol-rich lipid rafts as a platform for SARS-CoV-2 entry, where receptors such as the angiotensin-converting enzyme-2 (ACE-2), heparan sulfate proteoglycans (HSPGs), human Toll-like receptors (TLRs), transmembrane serine proteases (TMPRSS), CD-147 and HDL-scavenger receptor B type 1 (SR-B1) are recruited for their interaction with the viral spike protein. FDA-approved drugs such as statins, metformin, hydroxychloroquine, and cyclodextrins (methyl-β-cyclodextrin) can disrupt cholesterol-rich lipid rafts to regulate key molecules in the immune signaling pathways triggered by SARS-CoV-2 infection. Taken together, better knowledge on cholesterol-rich lipid rafts in the SARS-CoV-2-host interactions will provide valuable insights into pathogenesis and the identification of novel therapeutic targets.

Monosialoganglioside GM1 Deficiency Inhibits the Neurotrophic Effects of GDNF by Disrupting Lipid Raft Assembly

Recent studies have shown that monosialoganglioside GM1 deficiency can inhibit the signal transduction process of glial cell line-derived neurotrophic factor (GDNF), which plays an important role in the pathogenesis of Parkinson's disease (PD). However, its specific mechanism still needs to be explored. We inhibited the expression of GM1 by treating cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). CCK-8 assay, EdU cell proliferation assay and Western blot assay were used to evaluate the effect of GM1 deficiency on the proliferation and differentiation of SH-SY5Y cells induced by GDNF and on the GDNF-RET signaling pathway. Lipid rafts were isolated by Triton X-100 solubilization and OptiPrepTM density gradient centrifugation. The alterations of lipid raft assembly and the translocation of RET into lipid rafts were evaluated after PDMP treatment. We found that PDMP treatment inhibited the proliferation and differentiation of SH-SY5Y cells induced by GDNF and reduced the phosphorylation of RET and its downstream signaling molecules Erk and Akt. In addition, after PDMP treatment, caveolin-1 and flotillin-1, the prototypical markers of lipid rafts, diffused from lipid rafts to non-lipid raft microdomains, and GDNF-induced RET translocation into lipid rafts was also reduced. These alterations could be partially reversed by adding exogenous GM1. Our results suggest that ganglioside GM1 deficiency could compromise the neurotrophic effects and signals downstream of GDNF by altering the assembly of lipid raft membrane microdomains.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

EGFR transactivates RON to drive oncogenic crosstalk

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

Mutation in OsFWL7 Affects Cadmium and Micronutrient Metal Accumulation in Rice

Micronutrient metals, such as Mn, Cu, Fe, and Zn, are essential heavy metals for plant growth and development, while Cd is a nonessential heavy metal that is highly toxic to both plants and humans. Our understanding of the molecular mechanisms underlying Cd and micronutrient metal accumulation in plants remains incomplete. Here, we show that OsFWL7, an FW2.2-like (FWL) family gene in Oryza sativa, is preferentially expressed in the root and encodes a protein localized to the cell membrane. The osfwl7 mutation reduces both the uptake and the root-to-shoot translocation of Cd in rice plants. Additionally, the accumulation of micronutrient metals, including Mn, Cu, and Fe, was lower in osfwl7 mutants than in the wildtype plants under normal growth conditions. Moreover, the osfwl7 mutation affects the expression of several heavy metal transporter genes. Protein interaction analyses reveal that rice FWL proteins interact with themselves and one another, and with several membrane microdomain marker proteins. Our results suggest that OsFWL7 is involved in Cd and micronutrient metal accumulation in rice. Additionally, rice FWL proteins may form oligomers and some of them may be located in membrane microdomains.

Biochemical and Cytological Interactions Between Callose Synthase and Microtubules in the Tobacco Pollen Tube

Callose is a cell wall polysaccharide involved in several fundamental biological processes, ranging from plant development to response to abiotic and biotic stresses. To understand how callose deposition is regulated, it is important to know how its synthesizing enzyme, i.e., callose synthase, is regulated and if it interacts with vesicular-cytoskeletal system of plant cells. Actin filaments are thought to determine the long-range distribution of callose synthase through transport vesicles. Unlike other enzymes (such as cellulose synthase) that synthesize cell wall polysaccharides, the spatial and biochemical relationships between callose synthase and microtubules are poorly understood. Some experimental evidence already support the association between callose synthase and tubulin, however, despite its importance in maintaining plant integrity, knowledge about regulation of callose biosynthesis is still limited. Here we investigated the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in a model system, pollen tube, where callose is an essential cell wall component. Native 2-D electrophoresis and isolation of the callose synthase complex confirmed that callose synthase is associated with tubulin and can interface with cortical microtubules. In contrast, actin and sucrose synthase (which supplies UDP-glucose to callose synthase) are not permanently associated with callose synthase. Immunogold labeling showed strong colocalization of the enzyme and microtubules; this association is occasionally mediated by vesicles. The association between callose synthase and vesicles was also demonstrated by co-distribution between the enzyme and Rab11b; in addition, the not homogeneous distribution of callose synthase in cell membranes is also shown by analysis of membrane microdomains.

Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

A novel mechanism of thrombocytopenia by PS exposure through TMEM16F in sphingomyelin synthase 1 deficiency

Sphingomyelin synthase 1 (SMS1) contributes to the generation of membrane sphingomyelin (SM) and affects SM-mediated physiological functions. Here, we describe the hematologic phenotypes, such as reduced circulating platelets and dysfunctional hemostasis, in SMS1-deficient (SMS1-KO) mice. SMS1-KO mice display pathologic manifestations related to idiopathic thrombocytopenia (ITP), including relatively high amounts of peripheral blood reticulated platelets, enhanced megakaryopoiesis in the bone marrow and spleen, and splenomegaly. Deficiency of SMS1, but not SMS2, prevented SM production and enhanced phosphatidylserine (PS) externalization on the plasma membranes of platelets and megakaryocytes. Consequently, SMS1-KO platelets were excessively cleared by macrophages in the spleen. Multimer formation in the plasma membrane of TMEM16F, a known calcium (Ca2+)-activated nonselective ion channel and Ca2+-dependent PS scramblase, was enhanced; the result was PS externalization to outer leaflets through increased Ca2+ influx in immortalized mouse embryonic fibroblasts established from SMS1-KO mice (SMS1-KO tMEFs), as seen with SMS1-KO platelets. Thus, SMS1 deficiency changed the TMEM16F distribution on the membrane microdomain, regulating Ca2+ influx-dependent PS exposure. SMS1-KO tMEFs in which TMEM16F was knocked out by using the CRISPR/Cas9 system lacked both the Ca2+ influx and excess PS exposure seen in SMS1-KO tMEFs. Therefore, SM depletion on platelet membrane microdomains due to SMS1 deficiency enhanced PS externalization via a Ca2+ influx through TMEM16F activation, leading to elevated platelet clearance and causing hemostasis dysfunction through thrombocytopenia. Our current findings show that the SM-rich microdomain generated by SMS1 is a potent regulator of thrombocytopenia through TMEM16F, suggesting that its dysfunction may be a novel additional mechanism of ITP.

Membrane lipid raft homeostasis is directly linked to neurodegeneration

Age-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD) are an unmet health need, with significant economic and societal implications, and an ever-increasing prevalence. Membrane lipid rafts (MLRs) are specialised plasma membrane microdomains that provide a platform for intracellular trafficking and signal transduction, particularly within neurons. Dysregulation of MLRs leads to disruption of neurotrophic signalling and excessive apoptosis which mirrors the final common pathway for neuronal death in ALS, PD and AD. Sphingomyelinase (SMase) and phospholipase (PL) enzymes process components of MLRs and therefore play central roles in MLR homeostasis and in neurotrophic signalling. We review the literature linking SMase and PL enzymes to ALS, AD and PD with particular attention to attractive therapeutic targets, where functional manipulation has been successful in preclinical studies. We propose that dysfunction of these enzymes is upstream in the pathogenesis of neurodegenerative diseases and to support this we provide new evidence that ALS risk genes are enriched with genes involved in ceramide metabolism (P=0.019, OR = 2.54, Fisher exact test). Ceramide is a product of SMase action upon sphingomyelin within MLRs, and it also has a role as a second messenger in intracellular signalling pathways important for neuronal survival. Genetic risk is necessarily upstream in a late age of onset disease such as ALS. We propose that manipulation of MLR structure and function should be a focus of future translational research seeking to ameliorate neurodegenerative disorders.