Samuel Lara-González

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an α/β-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.

Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export.

XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.

Structure and biochemical characterization of proliferating cellular nuclear antigen from a parasitic protozoon.

Proliferating cellular nuclear antigen (PCNA) is a toroidal-shaped protein that is involved in cell-cycle control, DNA replication and DNA repair. Parasitic protozoa are early-diverged eukaryotes that are responsible for neglected diseases. In this work, a PCNA from a parasitic protozoon was identified, cloned and biochemically characterized and its crystal structure was determined. Structural and biochemical studies demonstrate that PCNA from Entamoeba histolytica assembles as a homotrimer that is able to interact with and stimulate the activity of a PCNA-interacting peptide-motif protein from E. histolytica, EhDNAligI. The data indicate a conservation of the biochemical mechanisms of PCNA-mediated interactions between metazoa, yeast and parasitic protozoa.

Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight β-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.

Structural and thermodynamic folding characterization of triosephosphate isomerases from Trichomonas vaginalis reveals the role of destabilizing mutations following gene duplication

We report the structures and thermodynamic analysis of the unfolding of two triosephosphate isomerases (TvTIM1 and TvTIM2) from Trichomonas vaginalis. Both isoforms differ by the character of four amino acids: E/Q 18, I/V 24, I/V 45, and P/A 239. Despite the high sequence and structural similarities between both isoforms, they display substantial differences in their stabilities. TvTIM1 (E18, I24, I45, and P239) is more stable and less dissociable than TvTIM2 (Q18, V24, V45, and A239). We postulate that the identities of residues 24 and 45 are responsible for the differences in monomer stability and dimer dissociability, respectively. The structural difference between both amino acids is one methyl group. In TvTIMs, residue 24 is involved in packing α‐helix 1 against α‐helix 2 of each monomer and residue 45 is located at the center of the dimer interface forming a “ball and socket” interplay with a hydrophobic cavity. The mutation of valine at position 45 for an alanine in TvTIM2 produces a protein that migrates as a monomer by gel filtration. A comparison with known TIM structures indicates that this kind of interplay is a conserved feature that stabilizes dimeric TIM structures. In addition, TvTIMs are located in the cytoplasm and in the membrane. As TvTIM2 is an easily dissociable dimer, the dual localization of TvTIMs may be related to the acquisition of a moonlighting activity of monomeric TvTIM2. To our knowledge, this is the simplest example of how a single amino acid substitution can provide alternative function to a TIM barrel protein. Proteins 2014; 82:22–33.

Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

Archetypical and Specialized DNA Replication Proteins in Entamoeba histolytica

Accurate DNA replication and repair are essential tasks for survival. In eukaryotes, DNA polymerases replicate genomes that can be composed of billions of base pairs. These genomes can be chemically damaged or modified, jeopardizing its integrity, and cells have evolved mechanisms to ameliorate the mutagenic effect of DNA damage. DNA replication and DNA lesion bypass in bacteria, yeast, and humans have been widely studied; however, little is known about these processes in other organisms. Entamoeba histolytica is a parasitic protozoan responsible for amebic dysentery and hepatic abscess. Herein, we define the DNA replication apparatus of Entamoeba histolytica and review the biochemical peculiarities of family A and family B2 DNA polymerases involved in DNA lesion bypass. Our data indicate that E. histolytica is a mosaic of archetypical family B DNA polymerases (α, e, and δ) present at the replication fork and specialized DNA polymerases with novel lesion bypass properties.