The immune cell infiltrate in the tumour microenvironment of phaeochromocytomas and paragangliomas.

Emerging evidence suggests the composition of the tumour microenvironment (TME) correlates with clinical outcome and that each tumour type has a unique TME including a variable population of inflammatory cells. We performed immunohistochemistry on 65 phaeochromocytoma and paraganglioma (PPGL) tumour samples with 20 normal adrenal medulla samples for comparison. The immune cells assessed were macrophages, lymphocytes and neutrophils, and we compared the proportion of infiltration of these immune cells with clinical and histopathological factors. There was a higher proportion of immune cells in tumour tissue compared to non-neoplastic adrenal medulla tissue, with a predominance of macrophages. There was a higher proportion of M2:M1 macrophages and T-helper lymphocytes in aggressive tumours compared to indolent ones. For SDHB-associated tumours, there was a higher proportion of M2 macrophage infiltration, with higher M2:M1 in aggressive SDHB PPGLs compared to indolent tumours. These data demonstrate that immune cells do infiltrate the TME of PPGLs, confirming that PPGLs are immunologically active tumours. Differences in the TME of PPGLs were observed between aggressive and indolent tumours. These differences could potentially be exploited as an aid in predicting tumour behaviour.

The majority of adrenocorticotropin receptor (melanocortin 2 receptor) mutations found in familial glucocorticoid deficiency type 1 lead to defective trafficking of the receptor to the cell surface.

CONTEXT: There are at least 24 missense, nonconservative mutations found in the ACTH receptor [melanocortin 2 receptor (MC2R)] that have been associated with the autosomal recessive disease familial glucocorticoid deficiency (FGD) type 1. The characterization of these mutations has been hindered by difficulties in establishing a functional heterologous cell transfection system for MC2R. Recently, the melanocortin 2 receptor accessory protein (MRAP) was identified as essential for the trafficking of MC2R to the cell surface; therefore, a functional characterization of MC2R mutations is now possible. OBJECTIVE: Our objective was to elucidate the molecular mechanisms responsible for defective MC2R function in FGD. METHODS: Stable cell lines expressing human MRAPalpha were established and transiently transfected with wild-type or mutant MC2R. Functional characterization of mutant MC2R was performed using a cell surface expression assay, a cAMP reporter assay, confocal microscopy, and coimmunoprecipitation of MRAPalpha. RESULTS: Two thirds of all MC2R mutations had a significant reduction in cell surface trafficking, even though MRAPalpha interacted with all mutants. Analysis of those mutant receptors that reached the cell surface indicated that four of six failed to signal, after stimulation with ACTH. CONCLUSION: The majority of MC2R mutations found in FGD fail to function because they fail to traffic to the cell surface.

Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.

We studied the ability of heat shock, DnaJ-like-1 (HSJ1) proteins (which contain DnaJ and ubiquitin-interacting motifs) to reduce polyglutamine-mediated inclusion formation. The experiments demonstrated that expression of heat shock protein 70 (hsp70), hsp40, HSJ1a, and HSJ1b significantly reduced protein inclusion formation in a model of spinal and bulbar muscular atrophy (SBMA). HSJ1a also mediated a significant decrease in the number of inclusions formed in a primary neuronal model of protein aggregation. Studies to elucidate the mechanisms underlying these reductions showed that hsp70 and hsp40 increased chaperone-mediated refolding. In contrast, expression of HSJ1 proteins did not promote chaperone activity but caused an increase in ubiquitylation. Furthermore, HSJ1a was associated with a ubiquitylated luciferase complex, and in the presence of HSJ1a but not an HSJ1a UIM mutant (HSJ1a-deltaUIM) there was a reduction in luciferase protein levels. Together these results show that HSJ1 proteins mediated an increase in target protein degradation via the ubiquitin-proteasome system (UPS). We also found that the expression of HSJ1a significantly decreased the number of neurons containing inclusions in an in vivo model of polyglutamine disease. These findings indicate that targeted modification of the UPS to facilitate degradation of misfolded proteins may represent a highly effective therapeutic avenue for the treatment of polyglutamine disease.

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK.

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.

Identification and characterization of a human mitochondrial homologue of the bacterial co-chaperone GrpE.

We have identified a novel human cDNA with a predicted protein sequence that has 28% amino acid identity with the E. coli Hsp70 co-chaperone GrpE and designated it HMGE. Even with this low level of amino acid identity the human sequence could be efficiently modelled on the X-ray structure of the E. coli protein, suggesting that there may be significant functional conservation. Indeed, HMGE expressed in E. coli as a GST fusion protein co-purified with the E. coli Hsp70 protein DnaK in the absence of ATP. DnaK could be released from the GST-HMGE with a Mg-ATP wash. Subcellular fractionation and immunocytochemistry studies using antisera raized against HMGE show that it is a mitochondrial protein. In contrast to studies of rat GrpE, however, HMGE also appears to bind the constitutive cytosolic Hsp70, Hsc70, in addition to mitochondrial Hsp70, Mt-Hsp70. We have previously shown that Hsc70 nucleotide-exchange is rate limiting in the presence of the DnaJ-protein, HSJ1b. However, HMGE was found to inhibit the HSJ1b-enhanced Hsc70 ATPase activity and may mediate this inhibition by binding the DnaJ-protein, HSJ1b. This is the first description of a direct interaction between a DnaJ protein and GrpE-like protein. These studies suggest that the structure of GrpE has been conserved throughout evolution and that the conserved structure can interact with several forms of Hsp70, but that HMGE cannot form part of the reaction cycle for cytosolic Hsc70.

Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane.

The X-linked retinitis pigmentosa (XLRP) gene, RP2, codes for a novel 350 amino acid protein of unknown function. We have identified putative sites for N-terminal acyl modification by myristoylation and palmitoylation in the RP2 protein. The RP2 protein is expressed ubiquitously in human tissues at relatively low levels (0.01% of total protein) and has a predominantly plasma membrane localization in cultured cells, as would be expected if the protein was subject to dual N-terminal acylation. Furthermore, mutagenesis of residues potentially required for N-terminal acylation prevents targeting of RP2 to the plasma membrane and the N-terminal 15 amino acids of the protein appear to be sufficient for this targeting. Our data suggest that the protein is dually acylated and that the palmitoyl moiety is responsible for targeting of the myristoylated protein from intracellular membranes to the plasma membrane. The effect of two mutations, which have been reported as causes of XLRP, R118H and DeltaS6, were investigated. The R118H mutation does not affect the normal plasma membrane localization of RP2; in contrast, the DeltaS6 mutation interferes with the targeting of the protein to the plasma membrane. Therefore, the DeltaS6 mutation may cause XLRP because it prevents normal amounts of RP2 reaching the correct cellular locale, whereas the R118H mutation is in a region of the protein that is vital for another aspect of RP2 function in the retina.