Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A).

Introduction: Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). Methods and results: Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. Discussion: Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions.

Rab32/38-Dependent and -Independent Transport of Tyrosinase to Melanosomes in B16-F1 Melanoma Cells.

B16-F1 melanoma cells have often been used as a model to investigate melanogenesis, but the evidence that melanosome biogenesis and transport occur by the same mechanisms in normal melanocytes and B16-F1 cells is insufficient. In this study, we established knockout B16-F1 cells for each of several key factors in melanogenesis, i.e., tyrosinase (Tyr), Hps4, Rab27A, and Rab32·Rab38 (Rab32/38), and then compared their phenotypes with the phenotypes of corresponding mutant mouse melanocyte cell lines, i.e., melan-c, melan-le, melan-ash, and Rab32-deficient melan-cht cells, respectively. The results showed that Tyr and Rab27A are also indispensable for melanin synthesis and peripheral melanosome distribution, respectively, in B16-F1 cells, but that Hps4 or its downstream targets Rab32/38 are not essential for Tyr transport in B16-F1 cells, suggesting the existence of a Rab32/38-independent Tyr transport mechanism in B16-F1 cells. We then performed comprehensive knockdown screening of Rab small GTPases and identified Rab10 and Rab24, previously uncharacterized Rabs in melanocytes, as being involved in Tyr transport under Rab32/38-null conditions. Our findings indicate a difference between the Tyr transport mechanism in melanocytes and B16-F1 cells in terms of Rab32/38-dependency and a limitation in regard to using melanoma cells as a model for melanocytes, especially when investigating the mechanism of endosomal Tyr transport.

Large Rab GTPase Rab44 regulates microtubule-dependent retrograde melanosome transport in melanocytes.

Melanosomes are melanin-containing organelles in melanocytes, and they are responsible for skin and hair pigmentation in mammals. The intracellular distribution of melanosomes is mainly determined by the balance between their anterograde transport on actin filaments and retrograde transport on microtubules. Although we have shown previously that melanoregulin and Rab36 serve as cargo receptors on melanosomes for retrograde transport, their knockdown does not completely inhibit retrograde melanosome transport, suggesting the existence of an additional cargo receptor(s) in melanocytes. In this study, we investigated the possible involvement of an atypical large Rab, Rab44, which also contains EF-hand domains and a coiled-coil domain, in retrograde melanosome transport in mouse melanocytes (Rab27A-deficient melan-ash cells). Our results showed that Rab44 localizes on mature melanosomes through lipidation of its C-terminal Rab-like GTPase domain, and that its knockdown results in suppression of retrograde melanosome transport. In addition, our biochemical analysis indicated that Rab44 interacts with the dynein-dynactin motor complex via its coiled-coil domain-containing middle region. Since simultaneous depletion of Rab44, melanoregulin, and Rab36 resulted in almost complete inhibition of retrograde melanosome transport, we propose that Rab44 is the third cargo receptor. We also showed that the N-terminal region of Rab44, which contains EF-hand domains, is required for both retrograde melanosome transport and its Ca2+-modulated activities. Our findings indicated that Rab44 is a third melanosomal cargo receptor, and that, unlike other cargo receptors previously described, its transport function is regulated by Ca2+.

Compositional dependence of structures and hydrogen evolution reaction activity of platinum-group-metal quinary RuRhPdIrPt alloy nanoparticles.

Platinum-group-metal quinary RuRhPdIrPt alloy nanoparticles were synthesised with compositions slightly away from equimolar, and their crystal and electronic structures were investigated. Their lattice constant changed linearly with composition, while the d-band centre changed nonlinearly. Their catalytic activities for the hydrogen evolution reaction were not correlated with their d-band centre.

Biochemical and structural insights into Rab12 interactions with RILP and its family members.

Alongside its biosynthetic functions, the small GTPase Rab12 negatively regulates mast cell (MC) exocytosis by its interaction with RILP to promote retrograde transport of the MC secretory granules. Given the role of Rab effectors in mediating Rab functions, in this study we used biochemical and in silico tools to decipher Rab12 interactions with its RILP family effectors. We show that Rab12 interacts with RILP, RILP-L1 and RILP-L2 independently of each other, whereby lysine-71, in mouse Rab12, is critical for Rab12 interactions with RILP-L1 or RILP-L2, but is dispensable for the binding of RILP. Focusing on RILP, and relying on molecular dynamics simulations, functional mutational analyses and peptide inhibition assays, we propose a model for the Rab12-RILP complex, consisting of a RILP homodimer and a single molecule of active Rab12, that interacts with the RILP homology domain (RHD) of one RILP monomer and a C-terminal threonine in the other monomer via its switch I and switch II regions. Mutational analyses of RILP RHD also demonstrate its involvement in the regulation of MC secretory granule transport. Jointly, our results provide structural and functional insights into the Rab12-RILP complex on the basis of which new tools could be generated for decoding Rab12 functions.

Polyphenol-Retaining Decaffeinated Cocoa Powder Obtained by Supercritical Carbon Dioxide Extraction and Its Antioxidant Activity.

Cocoa beans contain many functional ingredients such as theobromine and polyphenols, but also contain a relatively high amount of caffeine, which can negatively impact human health. It is therefore desirable to reduce caffeine levels in cocoa powder used to make chocolate or cocoa beverages while retaining functional ingredients. We have established conditions for supercritical carbon dioxide (SCCO2) extraction that remove 80.1% of the caffeine from cocoa powder while retaining theobromine (94.1%) and polyphenols (84.7%). The antioxidant activity of the decaffeinated cocoa powder (DCP) made with this optimized SCCO2 extraction method was 85.3% that of non-processed cocoa powder. The total procyanidin and total polyphenol concentrations of the DCPs resulting from various SCCO2 extractions showed a significant positive correlation with oxygen radical absorbance capacity (ORAC). The correlation coefficient between total polyphenols and ORAC was higher than that between total procyanidins and ORAC; thus, the concentration of total polyphenols might be a greater factor in the antioxidant activity of DCP. These results indicate that we could remove large quantities of caffeine from conventional high-cocoa products while retaining the functional benefits of high polyphenol content. This SCCO2 extraction method is expected to be applicable high-cocoa products, such as dark chocolate.

Functional involvement of Rab1A in microtubule-dependent anterograde melanosome transport in melanocytes.

Melanosomes are transported to the cell periphery of melanocytes by coordination between bidirectional microtubule-dependent movements and unidirectional actin-dependent movement. Although both the mechanism of the actin-dependent melanosome transport and the mechanism of the microtubule-dependent retrograde melanosome transport in mammalian skin melanocytes have already been determined, almost nothing is known about the mechanism of the microtubule-dependent anterograde melanosome transport. Small GTPase Rab proteins are common regulators of membrane traffic in all eukaryotes, and in this study we performed genome-wide screening for Rab proteins that are involved in anterograde melanosome transport by expressing 60 different constitutive active (and negative) mutants, and succeeded in identifying Rab1A, originally described as a Golgi-resident Rab, as a prime candidate. Endogenous Rab1A protein was found to be localized to mature melanosomes in melanocytes, and its functional ablation either by siRNA-mediated knockdown or by overexpression of a cytosolic form of Rab1A-GTPase-activating protein/TBC1D20 induced perinuclear melanosome aggregation. The results of time-lapse imaging further revealed that long-range anterograde melanosome movements were specifically suppressed in Rab1A-deficient melanocytes, whereas retrograde melanosome transport occurred normally. Taken together, these findings indicate that Rab1A is the first crucial component of the anterograde melanosome transport machinery to be identified in mammalian skin melanocytes.

Melanoregulin regulates retrograde melanosome transport through interaction with the RILP-p150Glued complex in melanocytes.

Melanoregulin (Mreg), a product of the dilute suppressor gene, has been implicated in the regulation of melanosome transport in mammalian epidermal melanocytes, given that Mreg deficiency was found to restore peripheral melanosome distribution from perinuclear melanosome aggregation in Rab27A-deficient melanocytes. However, the function of Mreg in melanosome transport has remained unclear. Here, we show that Mreg regulates microtubule-dependent retrograde melanosome transport through the dynein-dynactin motor complex. Mreg interacted with the C-terminal domain of Rab-interacting lysosomal protein (RILP) and formed a complex with RILP and p150(Glued) (also known as dynactin subunit 1, DCTN1), a component of the dynein-dynactin motor complex, in cultured cells. Overexpression of Mreg, RILP or both, in normal melanocytes induced perinuclear melanosome aggregation, whereas knockdown of Mreg or functional disruption of the dynein-dynactin motor complex restored peripheral melanosome distribution in Rab27A-deficient melanocytes. These findings reveal a new mechanism by which the dynein-dynactin motor complex recognizes Mreg on mature melanosomes through interaction with RILP and is involved in the centripetal movement of melanosomes.

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2.

Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.

Varp is a novel Rab32/38-binding protein that regulates Tyrp1 trafficking in melanocytes.

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.