Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4⌬608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion. INTRODUCTIONRab GTPases serve as important regulators of membrane transport in eukaryotic cells (Deneka et al., 2003b). Most rabs are ubiquitously expressed, but some have a more restricted distribution. For instance, rab27a is highly expressed in melanocytes and hematopoietic and other secretory cells (Tolmachova et al., 2004). Mutations causing loss of rab27a function in human result in defects of pigmentation (Bahadoran et al., 2001) and defects in the granule exocytosis pathway in cytotoxic T lymphocytes (CTLs) (Menasche et al., 2000). This rare autosomal recessive disease is called Griscelli syndrome type II (GS2) (Sanal et al., 2002).Mutations in the genes encoding myosin-Va and the rab27a effector melanophilin also cause a pigmentation phenotype in human (Pastural et al., 1997;Menasche et al., 2003) and mice. These observations led to the identification of a ternary complex consisting of rab27a/melanophilin/myosin-Va that is essential for the normal function of melanosomes (Wu et al., 2002). A related complex containing rab27a/myRIP/myoVIIa seems to be important for melanosome localization in retinal pigment epithelium (Amraoui et al., 2002) and secretory granules in the PC12 pheochromocytoma cell line (Desnos et al., 2003).Melanocytes and hematopoietic cells combine the functions of lysosomes and secretory granules into a hybrid organelle, the melanosome and secretory lysosome, respectively. Secretory lysosomes are particularly found in cells of the hematopoietic lineage, such as natural killer cells, CTLs, mast cells, dendritic cells, B cells, and neutrophils. They have an acidic lumenal pH and contain lysosomal enzymes. Ne...

Tyrosinase is a copper-containing enzyme that regulates melanin biosynthesis in mammals. Mutations at a single N-glycosylation sequon of tyrosinase have been reported to be responsible for oculocutaneous albinism type IA in humans, characterized by inactive tyrosinase and the total absence of pigmentation. To probe the role that each N-glycosylation site plays in the synthesis of biologically active tyrosinase, we analyzed the calnexin mediated folding of tyrosinase N-glycosylation mutants. We have determined that four of the six potential Nglycosylation sites, including that associated with albinism, are occupied. Analysis of the folding pathway and activity of 15 tyrosinase mutants lacking one or more of the occupied N-glycosylation sites shows that glycans at any two N-glycosylation sites are sufficient to interact with calnexin and give partial activity, but a specific pair of sites (Asn 86 and Asn 371 ) is required for full activity. The mutants with less than two N-glycosylation sites do not interact with calnexin and show a complete absence of enzyme activity. Copper analysis of selected mutants suggests that the observed partial activity is due to two populations with differential copper content. By correlating the degree of folding with the activity of tyrosinase, we propose a local folding mechanism for tyrosinase that can explain the mechanism of inactivation of tyrosinase N-glycosylation mutants found in certain pigmentation disorders.

Regression in melanoma is a frequent biological event of uncertain prognostic value as the lesion exhibits heterogeneous phenotypical features, both at the morphological and immunohistochemical level. In the present study, we examined the expression of tissue inhibitors of metalloproteinases (TIMP1, TIMP2 and TIMP3) in melanoma with regression. We specifically examined the expression levels of these TIMPs in regressed components (RC) and non-regressed components (NRC) of the tumor and compared their expression levels with those in non-regressed melanomas. We found that TIMP1 was overexpressed in the NRC of melanomas with partial regression (PR) compared with the NRC in melanomas with segmental regression (SR) (P=0.011). TIMP2 was overexpressed in the NRC of melanomas with PR compared with the NRC in melanomas with SR (PR/SR, P=0.009); or compared with the NRC in melanomas with simultaneous SR-PR (P=0.002); or compared with melanomas without regression (absence of regression) (P=0.037). Moreover, TIMP3 was overexpressed in the NRC of all melanomas with SR as compared to the RC component (P=0.007). Our findings on the differential expression of TIMP1, TIMP2 and TIMP3 in melanomas with regression support the hypothesis that the morphological differences identified in the melanoma regression spectrum may have a correlation with prognosis. This may explain the controversial findings within the literature concerning the biological and prognostic role of regression in melanoma.