Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-speci c function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant ndings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was con rmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/ stability of multiple chaperone client proteins could be perturbed by a de cient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identi ed. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our ndings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP

Rapid proteasomal degradation of mutant proteins is the primary mechanism leading to tumorigenesis in patients with missense AIP mutations

CONTEXT The pathogenic effect of AIP mutations (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. OBJECTIVE To analyze the mechanism/speed of protein turnover of wild-type (WT) and missense AIP variants, correlating protein half-life with clinical parameters. DESIGN Half-life and protein-protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed. SETTING Clinical academic research institution. PATIENTS Data was obtained from our cohort of pituitary adenoma patients and literature-reported cases. INTERVENTIONS Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. GST pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by co-immunoprecipitation and gene knockdown. Relevant clinical data was collected. MAIN OUTCOME MEASURES Half-life of WT and mutant AIP proteins and its correlation with clinical parameters. RESULTS Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7h). AIP variants were divided in stable proteins (median 77.7h [IQR 60.7-92.9]), and those with short (27h [21.6-28.7]) or very short (7.7h [5.6-10.5]) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r=0.411, P=0.002). The FBXO3-containing SCF complex was identified as the E3 ubiquitin-ligase recognizing AIP. CONCLUSIONS AIP is a stable protein, driven to ubiquitination by the SCF complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

We identified rare coding variants associated with Alzheimer’s disease (AD) in a 3-stage case-control study of 85,133 subjects. In stage 1, 34,174 samples were genotyped using a whole-exome microarray. In stage 2, we tested associated variants (P<1×10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, an additional 14,997 samples were used to test the most significant stage 2 associations (P<5×10-8) using imputed genotypes. We observed 3 novel genome-wide significant (GWS) AD associated non-synonymous variants; a protective variant in PLCG2 (rs72824905/p.P522R, P=5.38×10-10, OR=0.68, MAFcases=0.0059, MAFcontrols=0.0093), a risk variant in ABI3 (rs616338/p.S209F, P=4.56×10-10, OR=1.43, MAFcases=0.011, MAFcontrols=0.008), and a novel GWS variant in TREM2 (rs143332484/p.R62H, P=1.55×10-14, OR=1.67, MAFcases=0.0143, MAFcontrols=0.0089), a known AD susceptibility gene. These protein-coding changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified AD risk genes. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to AD development

ITC data for CDC37-BRAF interactions for paper: Recognition of BRAF by CDC37 and Re-evaluation of the Activation mechanism for the Class 2 BRAF-L597R Mutant

Data for paper published in Biomolecules June 2022  Isothermal Titration Calorimetry results for CDC37-BRAF interactions. dil in the filename donates the heat of dilution. Heats of dilution are in to buffer. Pairs with the same date donate a set of experiments. After the date the interacting partner proteins or small molecule is shown. Use Origin program to access the data files. Abstract: The kinome specific co-chaperone, CDC37, is responsible for delivering BRAF to the Hsp90 complex, where it is then translocated to the RAS complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognising the C-lobe of BRAF kinase domain, while the C-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF.  We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signalling in a clinical setting, particularly for class 2 BRAF mutations. </p

PyMol cartoon showing dimerization of AIP TPR-domain through crystal lattice contacts.

<p>(A), The AIP domains are in green and yellow. Amino acid residues are in magenta or cyan, hydrogen bonds as blue dotted lines, water molecules as red spheres and bound TOMM20 AQSLAEDDVE-peptide used in the crystallization in gold. However, only residues AEDDVE are visible in the structure. The TPR domains are symmetrically related and hydrogen bonding is shown in only one half of the figure. The cartoon shows that Arg 304 is hydrogen bounded directly to the neighboring TOMM20 bound peptide (gold). (B), PyMol cartoon showing a close up of the main interactions between Arg 304 and bound peptide used in the crystallization (AQLSLAED₃D₄VE) in panel A. However, only residues AED₃D₄VE are visible in the structure. (C), Co-immunoprecipitation of Flag-AIP and Myc-AIP in the presence of TOMM20 peptide (AQSLAEDDVE). The results show that Flag-AIP and Myc-AIP do not co-immunoprecipitate. M, molecular mass markers, with molecular mass indicated to the left of the panel; lane 1 and 5 AIP input (cleared lysate) protein; lane 2 and 6 are anti-Myc co-immunoprecipitation, lanes 3 and 7 are anti-Flag co-immunoprecipitations, while lanes 4 and 8 are IgG control. Lanes 1–4 (first gel) was blotted for Myc tag and lanes 5–8 (second gel) for Flag tag. The arrow indicates the position where the flag- and myc-tagged AIP runs (40 Kd). (D), The core interaction of the AIP dimerization interface shows that E192 is buried and shielded from solvent by Ala 312, Arg 188 and Trp 279.</p

PyMOL diagram showing binding interactions.

<p>(A) Interactions with HSP90β EDASRMEEVD peptide and (B), with TOMM20 AQSLAEDDVE peptide bound to the TPR domain of AIP. Peptide residues that where visible (SRMEEVD and AEDDVE) are shown in red as single letter code. Dotted blue lines represent hydrogen bonds and green, the amino acid residues involved; red-colored spheres, water molecules and yellow residues, residues solely in van der Waals contact. The structures were obtained at 2.0 (PDB, 4AIF) and 1.9 Å (PDB, 4APO), respectively. (C), Molecular switching in the TPR domain of AIP. The alternative conformations of Lys 266 allow selection of the Hsp90 MEEVD- (green) or TOMM20 EDDVE-motif (cyan). Dotted blue lines represent hydrogen bonds while red-colored spheres represent water molecules.</p

Sequence conservation of the Cα-7h of AIP.

<p>(A), sequence alignment showing conservation of amino acid residues. Ss, Salmo salar (NM_001140060.1); Dr, Danio rerio (NM_214712.1); Rn, Rattus norvegicus (NM_172327.2); Mm, Macaca mulatta (NM_001194313); Ca, Chlorocebus aethiops (O97628); Hs, Homo sapiens (FJ514478.1); Bt, Bos taurus (NM_183082.1), Xt, Xenopus (Silurana) tropicalis (NM_001102749.1) and Cc, Caligus clemensi (BT080130.1). (I313⁺), Ile 313 represents the last residue in the sequence that is involved in packing interactions of the TPR domain. Mutations associated with disease are indicated above the sequence. (* below the sequence), Amino acids at these positions are identical; (:), highly conserved (.) or conserved. Arg 304 of Human AIP is shown in red type face. Numbers above the sequence (positions 1 to 15) represent residue numbers of the helical wheel shown in panel B. (B), Helical wheel showing the position of identical and conserved residues form the alignment in panel A for the Cα-7h of AIP. Orange, non-polar; green, polar uncharged; pink, acidic and blue, basic amino-acid residues. (C), PyMol cartoon showing a hypothetical helix (residues beyond Arg 325) with the identical and highly conserved amino acid residues shown in panels A and B. Conserved residues on one side of the helix are shown in green and on the other in yellow. Residue numbers shown are those in panel B, while those in brackets are actual residue numbers in panel A. (D), The TPR-domain of the R304* mutant of AIP. Deletion of the terminal region of AIP (transparent helical region) allows chaperone binding but disrupts association with PDE4A5 and AhR.</p

PyMol diagram showing the conservation of residues on the surface of AIP TPR-domain.

<p>The most highly conserved residues line the cavity of the TPR domain in which the TPR-motif containing peptides bind to.</p