βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins

Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM barrel protein, have previously been found to contribute 4–6 kcal mol⁻¹ to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)₈ motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM barrel proteins.     

X-ray structure of human acid-beta-glucosidase,the defective enzyme in Gaucher disease

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ~200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α)₈ TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.     

Half-Barrels Derived from a (β/α)8 Barrel β-Glycosidase Undergo an Activation Process

The evolution of (β/α)₈ barrel proteins is currently thought to have involved the fusion of two (β/α)₄ half-barrels, thereby conferring stability on the protein structure. After the formation of a whole (β/α)₈ barrel, this structure could evolve and diverge to form fully active enzymes. Interestingly, we show here that isolated (β/α)₄ half-barrels derived from the N- and C-terminal domains of the β-glucosidase Sfβgly (Sfβgly-N: residues 1 to 265; Sfβgly-C: residues 266 to 509) undergo an activation process, which renders them catalytically active. The rate constants of the activation process were calculated to be 0.029 and 0.032 h^-1 for Sfβgly-N and Sfβgly-C, respectively. Moreover, the Sfβgly-N and Sfβgly-C activation processes were simultaneous with modifications in their initial structure, which reduced the exposure of their tryptophan residues. Importantly, this activation was also coincident with an increase in the sizes of Sfβgly-N and Sfβgly-C particles. These novel observations suggest that the change in catalytic activity associated with the transition from a half to whole (β/α)₈ barrel might also have driven such an evolutionary process.     

Vitamin A Metabolite,All-trans-retinoic Acid,Mediates Alternative Splicing of Protein Kinase C δVIII (PKCδVIII) Isoform via Splicing Factor SC35

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cδ (PKCδ), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCδ, PKCδVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCδVIII via utilization of a downstream 5′ splice site of exon 10 on PKCδ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCδVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCδVIII alternative splicing. To identify the cis-elements involved in 5′ splice site selection we cloned a minigene, which included PKCδ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5′ splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCδ minigene promoted selection of 5′ splice site II. Mutation of the SC35 binding site in the PKCδ minigene abolished RA-mediated utilization of 5′ splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCδ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCδ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCδVIII.     

From Discrete to Continuum Models of Three-Dimensional Deformations in Epithelial Sheets

Metamorphic proteins, including proteins with high levels of sequence identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% sequence identity adopt the same fold. Which topologies can be bridged by these highly identical sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high sequence identity (80 and 77%, respectively) with engineered variants of protein G (G_A, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial sequence modification. Furthermore, the MBP variant (80% identical to G_A) remained active. Thermodynamic analysis of numerous G_A and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.     

An exonic splicing silencer in the testes-specific DNA ligase III beta exon

Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation.     

Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon “SRKR”

BK Ca²⁺-activated K⁺ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca²⁺-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK₀ and BK_SRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK₀ and BK_SRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK₀, BK_SRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca²⁺ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca²⁺-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BK_SRKR channels were significantly reduced compared with BK₀, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.     

Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3

The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific α-subunit and common β-subunit (βc; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (β_IL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL-3 receptor (IL-3Rα) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length (“SP1” isoform) and a novel isoform (denoted “SP2”) lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) Rα isoform can direct mIL-3 binding to two distinct sites on the β_IL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of β_IL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu²³, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the β_IL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of β_IL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.     

Coordinated control of sensitivity by two splice variants of Gαo in retinal ON bipolar cells

The high sensitivity of scotopic vision depends on the efficient retinal processing of single photon responses generated by individual rod photoreceptors. At the first synapse in the mammalian retina, rod outputs are pooled by a rod “ON” bipolar cell, which uses a G-protein signaling cascade to enhance the fidelity of the single photon response under conditions where few rods absorb light. Here we show in mouse rod bipolar cells that both splice variants of the G_o α subunit, Gα_o1 and Gα_o2, mediate light responses under the control of mGluR6 receptors, and their coordinated action is critical for maximizing sensitivity. We found that the light response of rod bipolar cells was primarily mediated by Gα_o1, but the loss of Gα_o2 caused a reduction in the light sensitivity. This reduced sensitivity was not attributable to the reduction in the total number of G_o α subunits, or the altered balance of expression levels between the two splice variants. These results indicate that Gα_o1 and Gα_o2 both mediate a depolarizing light response in rod bipolar cells without occluding each other’s actions, suggesting they might act independently on a common effector. Thus, Gα_o2 plays a role in improving the sensitivity of rod bipolar cells through its action with Gα_o1. The coordinated action of two splice variants of a single Gα may represent a novel mechanism for the fine control of G-protein activity.     

Differences between α- and β-Chain Mutants of Human Haemoglobin and between α- and β-Thalassaemia. Possible Duplication of the α-Chain Gene

Human adult haemoglobin consists of two unlike pairs of polypeptide chains, and can be described as α₂β₂. Amino-acid substitutions in either of the two types of chain result in α- and β-chain variants. In thalassaemia, which causes a lowered production of haemoglobin, the α or the β chain can be affected, the result being α- or β-thalassaemia. There is a quantitative difference in the proportion of α- and β-chain variants to normal haemoglobin in the respective heterozygotes, and there is also a difference in the pattern of inheritance of α- and β-thalassaemia: these could possibly be explained by assuming that man has one gene for the β- and two for the α-chain.     

Crystal Structure of the Stress-Inducible Human Heat Shock Protein 70 Substrate-Binding Domain in Complex with Peptide Substrate

The HSP70 family of molecular chaperones function to maintain protein quality control and homeostasis. The major stress-induced form, HSP70 (also called HSP72 or HSPA1A) is considered an important anti-cancer drug target because it is constitutively overexpressed in a number of human cancers and promotes cancer cell survival. All HSP70 family members contain two functional domains: an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate-binding domain (SBD); the latter is subdivided into SBDα and SBDβ subdomains. The NBD and SBD structures of the bacterial ortholog, DnaK, have been characterized, but only the isolated NBD and SBDα segments of eukaryotic HSP70 proteins have been determined. Here we report the crystal structure of the substrate-bound human HSP70-SBD to 2 angstrom resolution. The overall fold of this SBD is similar to the corresponding domain in the substrate-bound DnaK structures, confirming a similar overall architecture of the orthologous bacterial and human HSP70 proteins. However, conformational differences are observed in the peptide-HSP70-SBD complex, particularly in the loop L_{α, β} that bridges SBDα to SBDβ, and the loop L_L,1 that connects the SBD and NBD. The interaction between the SBDα and SBDβ subdomains and the mode of substrate recognition is also different between DnaK and HSP70. This suggests that differences may exist in how different HSP70 proteins recognize their respective substrates. The high-resolution structure of the substrate-bound-HSP70-SBD complex provides a molecular platform for the rational design of small molecule compounds that preferentially target this C-terminal domain, in order to modulate human HSP70 function.     

Expression of splice variants of 1α-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D₃ (calcitriol) is the most active natural metabolite of Vitamin D₃. It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D₃-1α-hydroxylase (1α-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1α-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1α-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1α-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1α-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.     

Temperature-dependent splicing of beta-globin pre-mRNA

A T→G mutation at nucleotide 705 of human β-globin intron 2 creates an aberrant 5′ splice site and activates a cryptic 3′ splice site upstream. In consequence, the pre-mRNA is spliced via aberrant splice sites, despite the presence of the still functional correct sites. Surprisingly, when IVS2-705 HeLa or K562 cells were cultured at temperatures below 30°C, aberrant splicing was inhibited and correct splicing was restored. Similar temperature effects were seen for another β-globin pre-mRNA, IVS2-745, and in a construct in which a β-globin intron was inserted into a coding sequence of EGFP. Temperature-induced alternative splicing was affected by the nature of the internal aberrant splice sites flanking the correct sites and by exonic sequences. The results indicate that in the context of thalassemic splicing mutations and possibly in other alternatively spliced pre-mRNAs, temperature is one of the parameters that affect splice site selection.