LRBA participates in vesicular trafficking and receptor recycling

CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) is a protein receptor located in the plasmatic membrane of activated T cells. It functions to regulate homeostasis and peripheral immunological tolerance through the inhibition of T cell activation, competing with the costimulatory molecule CD28 for the specific ligation of CD80 and CD86, which are expressed on the surface of antigen-presenting cells ¹⁵. While CD28 resides mainly on the cell surface, CTLA-4 is located first in intracellular compartments such as Golgi apparatus, endosomes, secretory granules and lysosomes. The T cell receptor (TCR) stimulation promotes CTLA-4 trafficking from vesicular compartments to the cell membrane, however, once it is already in the membrane, this receptor is continuously endocytosed via clathrin-coated vesicles. After endocytosis, some CTLA-4 molecules are recycled to the cell membrane while others are rapidly degraded in lysosomal compartments ^16,17. CTLA-4 contains a small cytoplasmic tail with two tyrosine motifs at position Y201VKM and Y218FIP. Several intracellular proteins bind to the Y201VKM motif, including AP-1 and AP-2 (clathrin-associated adapter). AP-2 mediates the internalization of CTLA-4 from the cell surface to endosomes and lysosomal compartments whereas AP-1 regulates CTLA-4 trafficking from the Golgi apparatus to endosomal compartments and lysosomes for degradation ¹⁷. In a recent study, it was demonstrated that LRBA is necessary for the vesicular trafficking of CTLA-4 towards the plasma membrane ⁸. LRBA regulates the recycling of CTLA-4 from endosomes to the cell membrane supporting the maintenance of its intracellular levels for the immediate mobilization to the surface membrane, as this molecule is required. Thus, a deficiency of LRBA favors a rapid degradation of CTLA-4 in the lysosome compartments, with its concomitant decrease both intracellular and extracellularly ⁸. This work also demonstrated that LRBA interacts through its ConA-like- and the PH-BEACH domains with the cytoplasmic tail of CTLA-4, specifically with the Y201VKM motif. Finally, the silencing of AP-1, but not AP-2 partially rescues the CTLA-4 loss in the LRBA deficient cells. These results suggest that LRBA blocks the trafficking of CTLA-4 to lysosomes, competing with AP-1 for binding to its specific binding motif ⁸. Recent findings indicated that Foxp3 + CD4 + cells from LRBA-deficient mouse spleen cells also exhibit an intracellular decrease in CTLA-4 ¹⁸.

Other study that demonstrates the LRBA role in vesicular trafficking is related to the epidermal growth factor receptor (EGFR). Dominant-negative LRBA mutants decreases the expression and phosphorylation of EGFR ¹⁴. EGFR is a tyrosine kinase receptor that is expressed in a wide variety of tissues, interacting not only with EGF but also with TGF-β (Transforming growth factor-β), and other ligands. This induces the dimerization and trans-auto-phosphorylation of the receptor by recruiting proteins that activate signaling pathways necessary for the proliferation, differentiation, cell growth, migration and inhibition of apoptosis ¹⁹. Similar to CTLA-4 in lymphocytes, the EGFR expression on the cell surface is regulated by clathrin-mediated endocytosis, which leads either to its degradation via the lysosome compartment or its recycling to the cell surface ^20,21. Defects in the vesicular trafficking of this receptor result in an aberrant localization, which enhances signaling, allowing the development of cancer ²². Although it is still not known if LRBA and EGFR interact physically, it has been demonstrated that AP-2 facilitates endocytosis and trafficking of EGFR by the endocytic route ^3.

Finally, LRBA deficient patients exhibited a decrease in apoptosis mediated by the death receptor Fas, as compared to healthy donors ⁶. Fas receptor promotes apoptosis through the extrinsic pathway, after binding to Fas ligand (FasL). After this binding, a process of FasL internalization occur, similar to EGFR, which is crucial to trigger apoptosis ²⁴. To date, it is unknown whether LRBA-deficient patients exhibit alterations in the amount of FasL in the plasmatic membrane, but the increase in serum FasL in these patients could suggest that the LRBA deficiency may have an effect on FasL, similar to CTLA-4, regulating its trafficking either towards plasmatic membrane or lysosomes ²⁵.

These evidences suggest that LRBA might be involved in the vesicular trafficking of other receptors in lymphocytes. In LRBA deficient mice, defects have been reported in the phosphorylation of ERK1/2 (extracellular signal-regulated kinases) and AKT in natural killer (NK) cells stimulated with anti-NKG2D or anti-NKp46 antibodies, with a significant decrease in the production of IFN-gamma by these cells after stimulation ²⁶. Although this study did not determine the expression levels of these receptors in LRBA-deficient mice, there is some evidence that the exposure to specific ligands induces the degradation of NKG2D and DAP10, the intracellular adapter molecule of this receptor in lysosomes ²⁷. This regulation might be mediated by clathrin ²⁸. It is not known if LRBA is involved in the intracellular trafficking of these receptors in NK cells. Likewise, considering that the LRBA deficiency leads to defects in antibody responses, it would be also important to consider whether this molecule modulates the expression and function of other receptors whose expression is modulated by vesicular compartments, especially in regard to B cells activation ²⁹.

Despite these studies on the LRBA-mediated modulation of some cellular receptors, little is known about the intracellular pathways that this molecule employ to perform this function. This transport seems to be carried out by the recruitment of proteins in clathrin-coated vesicles, on which the receptors are removed to early endosomes and subsequently degraded by lysosomal enzymes or recycled to the cell surface ³⁰. AP-1 is considered necessary for this trafficking. The location of AP-1 in the Golgi apparatus and endosomes depends on its direct binding to Arf (ADP-ribosylation factor) -1 and specifically in recycling endosomes, to Arf-6, among other protein complexes ³¹. As already mentioned, LRBA inhibits the degradation of CTLA-4 in lysosomes by competition with AP-1, which suggest that this protein is necessary for mobilization to these compartments ⁸. However, the relationship of LRBA with the endosomal pathway is unclear. The recycling of receptors also depends on vesicular compartments, and the proteins this required, namely, Arf, enzymes such as phospholipase D and Rab proteins. Interestingly, a study conducted in Drosophila suggests a possible functional interaction between bchs (blue cheese), a member of the BEACH family, and Rab11 ³². These findings suggest that, as well as Rab11, Bchs could be involved in the regulation of vesicle trafficking.

Therefore, studies quantifying the expression of the mentioned receptors in LRBA deficient cells (either by silencing or from patients) are needed. It would also be useful to use advanced microscopy techniques to investigate the location of LRBA in other organelles involved in this type of trafficking. In addition, studies of co-immunoprecipitation between LRBA and vesicular trafficking proteins, the use of drugs that block this trafficking or the implementation of negative dominant ARFs, phospholipase D or Rab, could expand our knowledge about the interaction of LRBA with other vesicular trafficking molecules and thus determine its relationship with the membrane expression of receptors such as EGFR, Fas or the activating receptors of NK cells, B cells or others in immune cells.

LRBA is a molecule involved in autophagy

Autophagy is a mechanism of lysosomal degradation by which the cell destroys damaged organelles and microorganisms and helps to recycle macromolecules. In this process, a double membrane of isolation or phagophore is formed near to the target, which is then elongated, surrounding it, to later mature with the assimilation of a cytosolic charge forming the autophagosome. For the maturation to be effective, the autophagosome must be fused with early or late endosomes forming a structure called amphisome ^33,34. Finally, the mature autophagosomes fuses with lysosomes to proceed with the degradation of its content by lysosomal proteases and hydrolytic enzymes ³⁵.

Several of the genes homologous to LRBA are involved in autophagy. WDFY3 (WD and FYVE zinc finger domain containing protein 3) encodes a protein that colocalizes with autophagosome markers ^36,37. In addition, the PH-BEACH domain interacts with the p62 protein ³⁴. P62 is a protein located in the autophagosome formation sites and is associated with both LC3 (microtubule-associated protein 1A / 1B-light chain 3) and ubiquitinated proteins ^34,38,39. LC3 is a cytosolic protein that when conjugated with a phosphatidylethanolamine is recruited to the autophagosome membrane favoring the autophagic influx ³⁴. On the other hand, defects in the gene mauve (mv) in Drosophila, which is ortholog of LYST, affect the autophagosome function ⁴⁰.

In immortalized B cells from patients with LRBA deficiency, a significant reduction in the autophagy influx in response to starvation, together with an increase in the Golgi apparatus area, accumulation of autophagosomes and the presence of centrioles is observed, in comparison with those cells from healthy individual ². These results suggest that the autophagy influx is impaired in the LRBA deficiency, a finding that was demonstrated by a deficient fusion of autophagosome to lysosomes in immortalized B cells from LRBA-deficient patients subjected to starvation ². However, what are the possible functions of LRBA in the autophagy influx? Considering that both, the cell surface receptor trafficking and autophagy need endocytic vesicles, and that LRBA is likely located in these vesicles, a possible hypothesis would be that LRBA facilitates fusion between autophagosomes and the late endosomes, and therefore, the formation of the amphisome. One of the markers of late endosomes is Rab7 and this molecule is required for the formation of amphisomas in CHO cells transfected with the dominant negative mutant of Rab7, T22N ³³. Another hypothesis is that, in the same manner as the CTLA-4 vesicular trafficking, also in the context of autophagy in lymphocytes, LRBA would compete with clathrin adapter proteins, regulating this process. This since molecules such as clathrin and its adapter proteins are also important for the autophagic influx ^40-42. On the other hand, in silico tools have predicted that LRBA interacts with LC3 ¹³.

In an attempt to associate the defects in autophagy with the clinical characteristics found in LRBA deficiency individuals, it is important to bear in mind that one of the most common clinical manifestations among these individuals is the gastropathy, phenotypically expressed in many cases as Inflammatory Disease Intestinal (IBD) or IBD-like disease, a condition characterized by recurrent destructive inflammation in the intestinal tract ^3-5. An increase in the production of IL-1β in the interstitial mucosa has been implicated with uncontrolled inflammation in the course of this disease ⁴³. Macrophages derived from ATG16L1 (autophagy-related 16-like 1)-knockout mice present with a defect in autophagy and exhibit a high production of IL-1β after LPS stimulation ⁴⁴. Likewise, the polymorphism rs2241880 in ATG16L1 increased the production of IL-1β in peripheral blood mononuclear cells isolated from patients with IBD ⁴⁵. Autophagy can regulate the production of IL-1β through two pathways: in the absence of autophagy, defective mitochondria accumulate generating an increase in ROS (reactivating oxygen species) and releasing mitochondrial DNA into the cytoplasm, events that further activate the inflammosome, increasing the production of IL-1β ^46,47. On the other hand, the degradation of pro-IL-1β mediated by autophagy processes limits the substrate available for the inflammasome, thus regulating the activation of IL-1β ^48,49. Therefore, alterations in autophagy would impair this mechanism, increasing the levels of IL-1β and inducing inflammation. It is not known if the intestinal manifestations in LRBA deficient patients are mediated by an excessive activity of the inflammosome and exaggerated production of IL-1β.

Microscopy studies with LRBA deficient cells are required to study the intracellular distribution of molecules such as Rab7 and LC3 after the autophagic influx. It would also be interesting to investigate if LRBA interacts physically with any of the Rab molecules involved in the different types of autophagy, by means of co-immunoprecipitation assays. The role of LRBA in inflamosome activity could be addressed by measuring the production of IL-1β in LRBA deficient phagocytic or intestinal epithelial cells or by determining the activity of active auto-inflammosome by means of redistribution of ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), which is a protein that recruits the necessary caspases for the activation of the inflammosome.

LRBA participates in apoptosis.

In LRBA deficient patients’ immortalized B cells subjected to starvation, a significant increase in apoptosis is observed ². Additionally, in LRBA-knockdown HeLa cells exposed to ultraviolet light or staurosporine, a greater cleavage of PARP (Poly (ADP-ribose) polymerase) and caspase 3 are observed, events that are necessary to initiate the cascade of signaling that eventually leads to apoptosis ¹⁴.

Apoptosis is a type of programmed cell death to control development and growth. For long time, caspases have been considered as the proteases involved in the execution of the apoptosis program, and nowadays it is known that the partial destabilization of the lysosomal membrane, with the consequent release of hydrolytic enzymes, can initiate and execute apoptosis, byactivating caspases ^50,51. In germinal centers derived from tonsils, it is observed that B cell lysosomal destabilization contributes to apoptosis ⁵⁰. The induction of lysosomal damage in these cells resulted in the cleavage of caspase 8 and also of DNA, due to the increased activity of cathepsin B in the cytoplasm. Interestingly, it has been observed that p53 induces lysosomal destabilization ⁵² and the LRBA promoter is negatively regulated by p53 ¹⁴. p53 is a tumor suppressor gene that encodes a nuclear transcription factor, whose expression increases in response to damage, blocking the cell cycle either to facilitate DNA repair or to induce apoptosis ⁵². It is possible to hypothesize that, p53 would induce lysosomal destabilization in the absence of LRBA, and therefore, trigger apoptosis in lymphocytes. However, the mechanism by which p53 induces lysosomal destabilization or how LRBA protects cells from apoptosis is up to date unknown.

However, a complex crosstalk between autophagy and apoptosis exists, that is critical for cell fate. Under certain cellular conditions, autophagy can promote cell survival and prevent apoptosis ⁵³. For example, increased autophagy in cells deprived of nutrients or growth factors allows cell survival by inhibiting apoptosis. A suggested mechanism by which autophagy inhibits apoptosis is the sequestration of mitochondria damaged by the autophagosome, thus preventing the released mitochondrial cytochrome c to form a functional apoptosome in the cytoplasm ^53. However, in other conditions, autophagy may culminate in cell death, dependently or independently of apoptosis ⁵³. Additionally, there are several proteins that share a dual role in both apoptosis and autophagy ⁵³. These autophagic proteins positively or negatively regulate mitochondrial apoptosis directly or indirectly through the cleavage of proteases ⁵⁴. In the context of LRBA deficient cells, defective autophagy together with an increase in apoptosis has been demonstrated. It is not known if either alterations in the clearance of damaged mitochondria or an exaggerated cleavage of proteases are responsible for this cellular phenotype.

Therefore, it is necessary to evaluate the quantity and integrity of the lysosomes in LRBA deficient immune cells. In addition, the kinetics of lysosomal destabilization could be estimated in relation to the kinetics of other apoptotic parameters such as mitochondrial instability, caspase-3 activation and DNA cleavage.