Inicio CSIC

Artículos Científicos

Structures of the Gβ-CCT and PhLP1-Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

PNAS. 2015; 112 (8): 2413-2418.

Plimpton RL, Cuéllar J, Lai CW, Aoba T, Makaju A, Franklin S, Mathis AD, Prince JT, Carrascosa JL, Valpuesta JM, Willardson BM.

Blood. 2015; 125 (7)G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1).

Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1–Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1–Gβ dimer for assembly with Gγ.

This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.

Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors

Blood. 2015; 125 (7).

Gómez-Moutón C, Fischer T, Peregil RM, Jiménez-Baranda S, Stossel TP, Nakamura F, Mañes S.

Blood. 2015; 125 (7)Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare congenital immunodeficiency often caused by mutations in the last 10 to 19 C-terminal amino acids of CXCR4. These mutations impair CXCR4 internalization and increase responsiveness to CXCL12. The CXCR4 C-terminal domain (C-tail) also has a binding site for the actin-binding protein filamin A (FLNA); it is not known whether FLNA binds to WHIM CXCR4 mutants or whether this interaction is implicated in the hyperfunction of these receptors.

Here we show that, in addition to interacting with the CXCR4 C-tail, FLNA interacted with a region in the receptor third intracellular loop (ICL3) spanning amino acids 238 to 246. This interaction involved specific FLNA repeats and was sensitive to Rho kinase inhibition. Deletion of the 238-246 motif accelerated CXCL12-induced wild-type (WT) receptor endocytosis but enabled CXCL12-mediated endocytosis and normalized signaling by the WHIM-associated receptor CXCR4R334X. CXCL12 stimulation triggered CXCR4R334X internalization in FLNA-deficient M2 cells but not in the FLNA-expressing M2 subclone A7; this suggests a role for FLNA in stabilization of WHIM-like CXCR4 at the cell surface. FLNA increased β-arrestin2 binding to CXCR4R334X in vivo, which provides a molecular basis for FLNA-mediated hyperactivation of WHIM receptor signaling.

We propose that FLNA interaction with ICL3 is central for endocytosis and signaling of WT and WHIM-like CXCR4 receptors.

Attitude toward living kidney donation: differences between students from two spanish universities

Transplant Proc. 2015; 47 (1): 10-12.

Martínez-Alarcón L, Ramis G, Gómez-Laguna J, Quereda JJ, Herrero-Medrano JM, Mrowiec A, Mendonça L, López-Navas A, Ríos A.

Transplant Proc. 2015; 47 (1): 10-12Introduction: Due to the current deficit of organs for transplantation, living kidney related donations (LKRD) should be promoted. Veterinarians often hold decision-making positions in the public health care system, and therefore can influence public opinion about organ donation. The objective was to analyze the attitude of Spanish veterinary students toward LKRD because they may influence public opinion in the future, and to determine the factors that condition it.

Materials and Methods: The study was carried out among fifth-year veterinary science students from 2 southern and southeastern Spanish universities. The students' attitude toward LKRD was assessed using a psychosocial, anonymous, self-administered questionnaire. Statistics used were χ2 test and Student t test.

Results: Data from the southern Spain university included a response rate of 87%. The survey showed that 94% of respondents would donate a kidney to a relative who needed it. This attitude toward LKRD was more favorable in women (P < .001) and in those who had discussed the subject with their families (P = .003). Nevertheless, only 58% would accept a kidney from a family member. Data from the southeastern Spain university included a response rate of 97%: 97% of the respondents would donate a kidney to a relative who needed it. This attitude was not associated with any psychosocial variables. However, only 58% would accept a kidney from a relative. There are no differences between the 2 universities (P = .879).

Conclusions: Although the attitude of veterinary students from southern and southeastern Spain toward LKRD was very favorable and there are no differences between them, only 58% of the students would accept an organ from a relative.

Fungal Ku prevents permanent cell cycle arrest by suppressing DNA damage signaling at telomeres

Nucleic Acids Res. 2015;. pii: gkv082.

de Sena-Tomás C, Yu EY, Calzada A, Holloman WK, Lue NF, Pérez-Martín J.

Nucleic Acids Res. 2015;. pii: gkv082The Ku heterodimer serves in the initial step in repairing DNA double-strand breaks by the non-homologous end-joining pathway. Besides this key function, Ku also plays a role in other cellular processes including telomere maintenance. Inactivation of Ku can lead to DNA repair defects and telomere aberrations. In model organisms where Ku has been studied, inactivation can lead to DNA repair defects and telomere aberrations. In general Ku deficient mutants are viable, but a notable exception to this is human where Ku has been found to be essential.

Here we report that similar to the situation in human Ku is required for cell proliferation in the fungus Ustilago maydis. Using conditional strains for Ku expression, we found that cells arrest permanently in G2 phase when Ku expression is turned off. Arrest results from cell cycle checkpoint activation due to persistent signaling via the DNA damage response (DDR). Our results point to the telomeres as the most likely source of the DNA damage signal. Inactivation of the DDR makes the Ku complex dispensable for proliferation in this organism. Our findings suggest that in U. maydis, unprotected telomeres arising from Ku depletion are the source of the signal that activates the DDR leading to cell cycle arrest.

Irreversible covalent modification of type I dehydroquinase with a stable Schiff base

Org Biomol Chem. 2015; 13 (3): 706-716.

Tizón L, Maneiro M, Peón A, Otero JM, Lence E, Poza S, van Raaij MJ, Thompson P, Hawkins AR, González-Bello C.

Org Biomol Chem. 2015; 13 (3): 706-716The irreversible inhibition of type I dehydroquinase (DHQ1), the third enzyme of the shikimic acid pathway, is investigated by structural, biochemical and computational studies. Two epoxides, which are mimetics of the natural substrate, were designed as irreversible inhibitors of the DHQ1 enzyme and to study the binding requirements of the linkage to the enzyme. The epoxide with the S configuration caused the covalent modification of the protein whereas no reaction was obtained with its epimer.

The first crystal structure of DHQ1 from Salmonella typhi covalently modified by the S epoxide, which is reported at 1.4 Å, revealed that the modified ligand is surprisingly covalently attached to the essential Lys170 by the formation of a stable Schiff base. The experimental and molecular dynamics simulation studies reported here highlight the huge importance of the conformation of the C3 carbon of the ligand for covalent linkage to this type of aldolase I enzyme, revealed the key role played by the essential His143 as a Lewis acid in this process and show the need for a neatly closed active site for catalysis.