The conformation of tetraspanins CD53 and CD81 differentially affects their nanoscale organization and interaction with their partners.

Tetraspanins, including CD53 and CD81, are four-transmembrane proteins that affect the membrane organization to regulate cellular processes including migration, proliferation, and signaling. However, it is unclear how the organizing function of tetraspanins is regulated at the molecular level. Here, we investigated whether recently proposed "open" and "closed" conformations of tetraspanins regulate the nanoscale organization of the plasma membrane of B cells. We generated conformational mutants of CD53 (F44E) and CD81 (4A, E219Q) that represent the "closed" and "open" conformation, respectively. Surface expression of these CD53 and CD81 mutants was comparable to that of WT protein. Localization of mutant tetraspanins into nanodomains was visualized by super-resolution direct stochastic optical reconstruction microscopy. Whereas the size of these nanodomains was unaffected by conformation, the clustered fraction of "closed" CD53 was higher and of "open" CD81 lower than respective WT protein. In addition, KO cells lacking CD53 showed an increased likelihood of clustering of its partner CD45. Interestingly, "closed" CD53 interacted more with CD45 than WT CD53. Absence of CD81 lowered the cluster size of its partner CD19 and "closed" CD81 interacted less with CD19 than WT CD81, but "open" CD81 did not affect CD19 interaction. However, none of the tetraspanin conformations made significant impact on the nanoscale organization of their partners CD19 or CD45. Taken together, conformational mutations of CD53 and CD81 differentially affect their nanoscale organization, but not the organization of their partner proteins. This study improves the molecular insight into cell surface nanoscale organization by tetraspanins.

The tetraspanin transmembrane protein CD53 mediates dyslipidemia and integrates inflammatory and metabolic signaling in hepatocytes.

Tetraspanins are transmembrane signaling and proinflammatory proteins. Prior work demonstrates that the tetraspanin, CD53/TSPAN25/MOX44, mediates B-cell development and lymphocyte migration to lymph nodes and is implicated in various inflammatory diseases. However, CD53 is also expressed in highly metabolic tissues, including adipose and liver; yet its function outside the lymphoid compartment is not defined. Here, we show that CD53 demarcates the nutritional and inflammatory status of hepatocytes. High-fat exposure and inflammatory stimuli induced CD53 in vivo in liver and isolated primary hepatocytes. In contrast, restricting hepatocyte glucose flux through hepatocyte glucose transporter 8 deletion or through trehalose treatment blocked CD53 induction in fat- and fructose-exposed contexts. Furthermore, germline CD53 deletion in vivo blocked Western diet-induced dyslipidemia and hepatic inflammatory transcriptomic activation. Surprisingly, metabolic protection in CD53 KO mice was more pronounced in the presence of an inciting inflammatory event. CD53 deletion attenuated tumor necrosis factor alpha-induced and fatty acid + lipopolysaccharide-induced cytokine gene expression and hepatocyte triglyceride accumulation in isolated murine hepatocytes. In vivo, CD53 deletion in nonalcoholic steatohepatitis diet-fed mice blocked peripheral adipose accumulation and adipose inflammation, insulin tolerance, and liver lipid accumulation. We then defined a stabilized and trehalase-resistant trehalose polymer that blocks hepatocyte CD53 expression in basal and over-fed contexts. The data suggest that CD53 integrates inflammatory and metabolic signals in response to hepatocyte nutritional status and that CD53 blockade may provide a means by which to attenuate pathophysiology in diseases that integrate overnutrition and inflammation, such as nonalcoholic steatohepatitis and type 2 diabetes.

The tetraspanin CD53 protects stressed hematopoietic stem cells via promotion of DREAM complex-mediated quiescence.

The hematopoietic stem cell (HSC) cycle responds to inflammatory and other proliferative stressors; however, these cells must quickly return to quiescence to avoid exhaustion and maintain their functional integrity. The mechanisms that regulate this return to quiescence are not well understood. Here, we show that tetraspanin CD53 is markedly upregulated in HSCs in response to a variety of inflammatory and proliferative stimuli and that the loss of CD53 is associated with prolonged cycling and reduced HSC function in the context of inflammatory stress. Mechanistically, CD53 promotes the activity of the dimerization partner, RB-like, E2F, and multi-vulva class B (DREAM) transcriptional repressor complex, which downregulates genes associated with cycling and division. Proximity labeling and confocal fluorescence microscopy studies showed that CD53 interacts with DREAM-associated proteins, specifically promoting the interaction between Rbl2/p130 and its phosphatase protein phosphatase 2A (PP2A), effectively stabilizing p130 protein availability for DREAM binding. Together, these data identified a novel mechanism by which stressed HSCs resist cycling.

Tetraspanin CD53 modulates lymphocyte trafficking but not systemic autoimmunity in Lyn-deficient mice.

The leukocyte-restricted tetraspanin CD53 has been shown to promote lymphocyte homing to lymph nodes (LNs) and myeloid cell recruitment to acutely inflamed peripheral organs, and accelerate the onset of immune-mediated disease. However, its contribution in the setting of chronic systemic autoimmunity has not been investigated. We made use of the Lyn-/- autoimmune model, generating Cd53-/- Lyn-/- mice, and compared trafficking of immune cells into secondary lymphoid organs and systemic autoimmune disease development with mice lacking either gene alone. Consistent with previous observations, absence of CD53 led to reduced LN cellularity via reductions in both B and T cells, a phenotype also observed in Cd53-/- Lyn-/- mice. In some settings, Cd53-/- Lyn-/- lymphocytes showed greater loss of surface L-selectin and CD69 upregulation above that imparted by Lyn deficiency alone, indicating that absence of these two proteins can mediate additive effects in the immune system. Conversely, prototypical effects of Lyn deficiency including splenomegaly, plasma cell expansion, elevated serum immunoglobulin M and anti-nuclear antibodies were unaffected by CD53 deficiency. Furthermore, while Lyn-/- mice developed glomerular injury and showed elevated glomerular neutrophil retention above than that in wild-type mice, absence of CD53 in Lyn-/- mice did not alter these responses. Together, these findings demonstrate that while tetraspanin CD53 promotes lymphocyte trafficking into LNs independent of Lyn, it does not make an important contribution to development of autoimmunity, plasma cell dysfunction or glomerular injury in the Lyn-/- model of systemic autoimmunity.

Open conformation of tetraspanins shapes interaction partner networks on cell membranes.

Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen-deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1-EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre-B cells toward a chemokine that supports B-cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin-partner interactions to support myriad cellular processes.

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

Surfaceome interrogation using an RNA-seq approach highlights leukemia initiating cell biomarkers in an LMO2 T cell transgenic model.

The surfaceome is critical because surface proteins provide a gateway for internal signals and transfer of molecules into cells, and surfaceome differences can influence therapy response. We have used a surfaceome analysis method, based on comparing RNA-seq data between normal and abnormal cells (Surfaceome DataBase Mining or Surfaceome DBM), to identify sets of upregulated cell surface protein mRNAs in an LMO2-mediated T-ALL mouse model and corroborated by protein detection using antibodies. In this model the leukemia initiating cells (LICs) comprise pre-leukaemic, differentiation inhibited thymocytes allowing us to provide a profile of the LIC surfaceome in which GPR56, CD53 and CD59a are co-expressed with CD25. Implementation of cell surface interaction assays demonstrates fluid interaction of surface proteins and CD25 is only internalized when co-localized with other proteins. The Surfaceome DBM approach to analyse cancer cell surfaceomes is a way to find targetable surface biomarkers for clinical conditions where RNA-seq data from normal and abnormal cell are available.

Neural Mobilization Treatment Decreases Glial Cells and Brain-Derived Neurotrophic Factor Expression in the Central Nervous System in Rats with Neuropathic Pain Induced by CCI in Rats.

Background. Glial cells are implicated in the development of chronic pain and brain-derived neurotropic factor (BDNF) released from activated microglia contributes to the nociceptive transmission. Neural mobilization (NM) technique is a method clinically effective in reducing pain sensitivity. Here we examined the involvement of glial cells and BDNF expression in the thalamus and midbrain after NM treatment in rats with chronic constriction injury (CCI). CCI was induced and rats were subsequently submitted to 10 sessions of NM, every other day, beginning 14 days after CCI. Thalamus and midbrain were analyzed for glial fibrillary acidic protein (GFAP), microglial cell OX-42, and BDNF using Immunohistochemistry and Western blot assays. Results. Thalamus and midbrain of CCI group showed increases in GFAP, OX-42, and BDNF expression compared with control group and, in contrast, showed decreases in GFAP, OX-42, and BDNF after NM when compared with CCI group. The decreased immunoreactivity for GFAP, OX-42, and BDNF in ventral posterolateral nucleus in thalamus and the periaqueductal gray in midbrain was shown by immunohistochemistry. Conclusions. These findings may improve the knowledge about the involvement of astrocytes, microglia, and BDNF in the chronic pain and show that NM treatment, which alleviates neuropathic pain, affects glial cells and BDNF expression.

Down-regulation of CD53 expression in Epinephelus coioides under LPS, poly (I:C), and cytokine stimulation.

Tetraspanins are a group of cell surface molecules involved in cell adhesion, motility, metastasis, signal transduction, and immune cell activation. Members of the tetraspanin family include CD9, CD37, CD63, CD53, and others. However, few tetraspanins have been investigated in teleosts. In this study, we obtained the open reading frame of CD53 cDNA from orange spotted grouper (Epinephelus coioices), an economically important fish. The predicted amino acid structure contains four membrane-spanning domains and a conserved CCG motif. The amino acid identity between human and grouper CD53 was only 38%; however, both CD53 proteins share the same structure. Quantitative real-time PCR revealed that mRNA is abundant in immune organs, including the head and trunk kidneys, spleen, thymus, gill, and blood. Immunochemistry and immunofluorescence analyses further revealed that CD53 was majorly expressed in the leukocytes of various organs. Finally, mRNA and protein expression for CD53 was down-regulated in fish treated with immune stimulators, including LPS, Poly (I:C), Vibrio, recombinant grouper IL-6, and CCL4. Our results indicate that the expression of CD53 may play important roles in pathogen invasion and inflammation reaction.

The tetraspanin web revisited by super-resolution microscopy.

The spatial organization of membrane proteins in the plasma membrane is critical for signal transduction, cell communication and membrane trafficking. Tetraspanins organize functional higher-order protein complexes called 'tetraspanin-enriched microdomains (TEMs)' via interactions with partner molecules and other tetraspanins. Still, the nanoscale organization of TEMs in native plasma membranes has not been resolved. Here, we elucidated the size, density and distribution of TEMs in the plasma membrane of human B cells and dendritic cells using dual color stimulated emission depletion (STED) microscopy. We demonstrate that tetraspanins form individual nanoclusters smaller than 120 nm and quantified that a single tetraspanin CD53 cluster contains less than ten CD53 molecules. CD53 and CD37 domains were adjacent to and displayed only minor overlap with clusters containing tetraspanins CD81 or CD82. Moreover, CD53 and CD81 were found in closer proximity to their partners MHC class II and CD19 than to other tetraspanins. Although these results indicate that tetraspanin domains are adjacently positioned in the plasma membrane, they challenge the current view of the tetraspanin web of multiple tetraspanin species organized into a single domain. This study increases the molecular understanding of TEMs at the nanoscale level which is essential for comprehending tetraspanin function in cell biology.

Inclusion of cocoa as a dietary supplement represses expression of inflammatory proteins in spinal trigeminal nucleus in response to chronic trigeminal nerve stimulation.

SCOPE: Central sensitization is implicated in the pathology of temporomandibular joint disorder and other types of orofacial pain. We investigated the effects of dietary cocoa on expression of proteins involved in the development of central sensitization in the spinal trigeminal nucleus (STN) in response to inflammatory stimulation of trigeminal nerves. METHODS AND RESULTS: Male Sprague-Dawley rats were fed either a control diet or an isocaloric diet consisting of 10% cocoa powder 14 days prior to bilateral injection of complete Freund's adjuvant (CFA) into the temporomandibular joint to promote prolonged activation of trigeminal ganglion neurons and glia. While dietary cocoa stimulated basal expression of glutamate-aspartate transporter and mitogen-activated protein kinase phosphatase-1 when compared to animals on a normal diet, cocoa suppressed basal calcitonin gene-related peptide levels in the STN. CFA-stimulated levels of protein kinase A, P2X3 , P-p38, glial fibrillary-associated protein, and OX-42, whose elevated levels in the STN are implicated in central sensitization, were repressed to near control levels in animals on a cocoa-enriched diet. Similarly, dietary cocoa repressed CFA-stimulated inflammatory cytokine expression. CONCLUSION: Based on our findings, we speculate that cocoa-enriched diets could be beneficial as a natural therapeutic option for temporomandibular joint disorder and other chronic orofacial pain conditions.

Comparative expression profiling identifies differential roles for Myogenin and p38α MAPK signaling in myogenesis.

Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling. However, the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown. Here, we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myogenesis. Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function. Furthermore, we found that in the absence of p38α signaling, Myog expression leads to the down-regulation of genes involved in cell cycle progression. Consistent with this, the expression of Myog is sufficient to induce cell cycle exit. Interestingly, p38α-defective, Myog-expressing myoblasts fail to form multinucleated myotubes, suggesting an important role for p38α in cell fusion. Through the analysis of p38α up-regulated genes, the tetraspanin CD53 was identified as a candidate fusion protein, a role confirmed both ex vivo in primary myoblasts, and in vivo during myofiber regeneration in mice. Thus, our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.

Integrated cross-species transcriptional network analysis of metastatic susceptibility.

Metastatic disease is the proximal cause of mortality for most cancers and remains a significant problem for the clinical management of neoplastic disease. Recent advances in global transcriptional analysis have enabled better prediction of individuals likely to progress to metastatic disease. However, minimal overlap between predictive signatures has precluded easy identification of key biological processes contributing to the prometastatic transcriptional state. To overcome this limitation, we have applied network analysis to two independent human breast cancer datasets and three different mouse populations developed for quantitative analysis of metastasis. Analysis of these datasets revealed that the gene membership of the networks is highly conserved within and between species, and that these networks predicted distant metastasis free survival. Furthermore these results suggest that susceptibility to metastatic disease is cell-autonomous in estrogen receptor-positive tumors and associated with the mitotic spindle checkpoint. In contrast, nontumor genetics and pathway activities-associated stromal biology are significant modifiers of the rate of metastatic spread of estrogen receptor-negative tumors. These results suggest that the application of network analysis across species may provide a robust method to identify key biological programs associated with human cancer progression.