Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms

Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5′-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.     

Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

VRK2 is a novel Ser-Thr kinase whose VRK2A isoform is located in the endoplasmic reticulum and mitochondrial membranes. We have studied the potential role that VRK2A has in the regulation of mitochondrial-mediated apoptosis. VRK2A can regulate the intrinsic apoptotic pathway in two different ways. The VRK2A protein directly interacts with Bcl-xL, but not with Bcl-2, Bax, Bad, PUMA or Binp-3L. VRK2A does not compete with Bax for interaction with Bcl-xL, and these proteins can form a complex that reduces apoptosis. Thus, high VRK2 levels confer protection against apoptosis. In addition, VRK2 knockdown results in an increased expression of BAX gene expression that is mediated by its proximal promoter, thus VRK2A behaves as a negative regulator of BAX. Low levels of VRK2A causes an increase in mitochondrial Bax protein level, leading to an increase in the release of cytochrome C and caspase activation, detected by PARP processing. VRK2A loss results in an increase in cell death that can be detected by an increase in annexinV+ cells. Low levels of VRK2A increase cell sensitivity to induction of apoptosis by chemotherapeutic drugs like camptothecin or doxorubicin. We conclude that VRK2A protein is a novel modulator of apoptosis.     

Genome Reduction Promotes Increase in Protein Functional Complexity in Bacteria

Obligate pathogenic and endosymbiotic bacteria typically experience gene loss due to functional redundancy, asexuality, and genetic drift. We hypothesize that reduced genomes increase their functional complexity through protein multitasking, in which many genes adopt new roles to counteract gene loss. Comparisons of interaction networks among six bacteria that have varied genome sizes (Mycoplasma pneumoniae, Treponema pallidum, Helicobacter pylori, Campylobacter jejuni, Synechocystis sp., and Mycobacterium tuberculosis) reveal that proteins in small genomes interact with proteins from a wider range of functions than do their orthologs in larger genomes. This suggests that surviving proteins form increasingly complex functional relationships to compensate for genes that are lost.     

Role of β-lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP-II

The β-lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein-protein interactions. BLIP and BLIP-II are structurally unrelated proteins that bind and inhibit TEM-1 β-lactamase. Both BLIPs share a common binding interface on TEM-1 and make contacts with many of the same TEM-1 surface residues. BLIP-II, however, binds TEM-1 over 150-fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM-1 amino acid residues that contact both BLIP and BLIP-II was examined by alanine mutagenesis and determination of the association (k_on) and dissociation (k_off) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP-II. This is consistent with the high affinity and small binding interface of the TEM-1-BLIP-II complex, which predicts per residue contributions should be higher for TEM-1 binding to BLIP-II versus BLIP. Two TEM-1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP-II with only M129 as a common hotspot for both. Thus, although the same TEM-1 surface binds to both BLIP and BLIP-II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed.     

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor

Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1–WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1–WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology.