Ana M. Rojas

The Nod-Like Receptor (NLR) Family: A Tale of Similarities and Differences

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs posses a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.

The Ras protein superfamily: Evolutionary tree and role of conserved amino acids

The Ras superfamily is a fascinating example of functional diversification in the context of a preserved structural framework and a prototypic GTP binding site. Thanks to the availability of complete genome sequences of species representing important evolutionary branch points, we have analyzed the composition and organization of this superfamily at a greater level than was previously possible. Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this protein superfamily sequence and the acquisition of distinct cellular functions. Together with advances in computational methods and structural studies, the sequence information has helped to identify features important for the recognition of molecular partners and the functional specialization of different members of the Ras superfamily.

Filamin-A regulates actin-dependent clustering of HIV receptors

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA–ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.

The amino acid transport system y+L/4F2hc is a heteromultimeric complex

4F2hc is an almost ubiquitous transmembrane protein in mammalian cells; upon expression in Xenopus laevis oocytes, it induces amino acid transport with characteristics of system y+L. Indirect evidence fostered speculation that function requires the association of 4F2hc with another protein endogenous to oocytes and native tissues. We show that expression of system y+L‐like amino acid transport activity by 4F2hc in oocytes is limited by an endogenous factor and that direct covalent modification of external cysteine residue(s) of an oocyte membrane protein blocks system y+ L/4F2hc transport activity, based on the following. 1) Induction of system y+L‐like activity saturates at very low doses of human 4F2hc cRNA (0.1 ng/oocyte). This saturation occurs with very low expression of 4F2hc at the oocyte surface, and further increased expression of the protein at the cell surface does not result in higher induction of system y+L‐like activity. 2) Human 4F2hc contains only two cysteine residues (C109 and C330). We mutated these residues, singly and in combination, to serine (C109S; CS1, C330S; CS2 and C109S‐C330S, Cys‐less). Mutation CS2 had no effect on the expressed system y+ L‐like transport activity, whereas C109S‐containing mutants (CS1 and Cys‐less) retained only partial y+L‐like transport activity (30 to 50% of wild type). 3) Hg2+, the organic mercury compounds pCMB, and the membrane‐impermeant p‐CMBS almost completely inactivated system y+L‐like induced by human 4F2hc wild type and all the mutants studied. This was reversed by β‐mercaptoethanol, indicating that external cysteine residue(s) are the target of this inactivation. 4) Sensitivity to Hg2+ inactivation is increased by pretreatment of oocytes with β‐mercaptoethanol or in the C109S‐containing mutants (CS1 and Cys‐less). The increased Hg2+ reactivity of C109S‐containing mutants supports the possibility that C109 may be linked by a disulfide bond to the Hg2+‐targeted cysteine residue of the associated protein. These results indicate that 4F2hc is intimately associated with a membrane oocyte protein for the expression of system y+L amino acid transport activity. To our knowledge, this is the first direct evidence for a heteromultimeric protein structure of an organic solute carrier in mammals.— Este´vez, R., Camps, M., Rojas, A. M., Testar, X., Deve ´s, R., Hediger, M. A., Zorzano, A., Palacı´n, M. The amino acid transport system y/L/4F2hc is a heteromultimeric complex. FASEB J. 12, 1319–1329 (1998)

Assessment of predictions submitted for the CASP7 function prediction category

Here we present a full overview of the Critical Assessment of Protein Structure Prediction (CASP7) function prediction category. Predictions were submitted for Gene Ontology molecular function terms, Enzyme Commission numbers, and ligand binding site residues. The first two categories were difficult to assess because very little new functional information becomes available after the experiment. The majority of the known Gene Ontology terms and all the Enzyme Commission numbers were available a priori to predictors before the experiment, so prediction for these two categories was not blind. Nevertheless, for Gene Ontology terms we were able to demonstrate that some groups made better predictions than others. In the binding residue category, the predictors did not know in advance which ligands were bound and therefore blind evaluation was possible, but there were disappointingly few predictions in this category. After CASP 6 and 7 the need to organize a more effective blind function prediction category is obvious, even if it means focusing on binding site prediction as the only category that can be truly assessed in the CASP spirit.This paper details the assessment process and evaluation results for the Critical Assessment of Protein Structure Prediction (CASP7) domain prediction category. Domain predictions were assessed using the Normalized Domain Overlap score introduced in CASP6 and the accuracy of prediction of domain break points. The results of the analysis clearly demonstrate that the best methods are able to make consistently reliable predictions when the target has a structural template, although they are less good when the domain break occurs in a region not covered by a template. The conditions of the experiment meant that it was impossible to draw any conclusions about domain prediction for free modeling targets and it was also difficult to draw many distinctions between the best groups. Two thirds of the targets submitted were single domains and hence regarded as easy to predict. Even those targets defined as having multiple domains always had at least one domain with a similar template structure. Proteins 2007.

The pseudo GTPase CENP-M drives human kinetochore assembly

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore–centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers. DOI: http://dx.doi.org/10.7554/eLife.02978.001

KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases

Potassium channel tetramerization domain (KCTD) proteins contain a bric‐a‐brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage‐gated potassium channels. Some BTB‐domain‐containing proteins have been shown recently to participate as substrate‐specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty‐two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post‐transcriptionally in peripheral blood lymphocytes stimulated through the T‐cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N‐terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate‐specific adaptor for cullin3‐based E3 ligases.

A Proteomic Characterization of Factors Enriched at Nascent DNA Molecules

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.

Gas1 Is Related to the Glial Cell-derived Neurotrophic Factor Family Receptors α and Regulates Ret Signaling

The growth arrest-specific gene 1 (Gas1) protein has been proposed to function during development as an inhibitor of growth and a mediator of cell death and is also re-expressed in adult neurons during excitotoxic insult. Here we have demonstrated that the Gas1 protein shows high structural similarity to the glial cell-derived neurotrophic factor (GDNF) family receptors α, which mediate GDNF responses through the receptor tyrosine kinase Ret. We found that Gas1 binds Ret in a ligand-independent manner and sequesters Ret in lipid rafts. Signaling downstream of Ret is thus modified through a mechanism that involves the adaptor protein Shc as well as ERK, eventually blocking Akt activation. Consequently, when Gas1 is induced, Ret-mediated GDNF-dependent survival effects are compromised.

The dual-specific protein tyrosine phosphatase family

Dual-specificity protein phosphatases (DSPs) belong to the protein tyrosine phosphatase (PTP) superfamily since they contain the conserved motif HCX2GX2R and share the same tertiary structure. The DSP family constitutes approximately half of all PTPs and includes a diverse group of proteins with a wide distribution among living organisms. They dephosphorylate proteins with phosphate on serine, threonine and/or tyrosine. The best-characterized substrates are the mitogenactivated protein kinases, which are dephosphorylated at tyrosine and threonine in a TXY motif. Additional substrates have been identified recently, ADF/cofilin for the slingshot DSP and glucokinase for DUSP12. DSPs can play key roles in multicellular organisms, as shown for the DSPs puckered and slingshot in the fruit fly and for LIP-1 in the worm. However, the physiological roles that DSPs play in higher vertebrates are largely unknown and there seems to be more redundancy. This chapter will discuss the evolution, structure, and function of the DSP family.