Mika Jormakka

Mutations in Alpha-Actinin-2 Cause Hypertrophic Cardiomyopathy: A Genome-Wide Analysis

OBJECTIVES This study describes a genome-wide linkage analysis of a large family with clinically heterogeneous hypertrophic cardiomyopathy (HCM). BACKGROUND Familial HCM is a disorder characterized by genetic heterogeneity. In as many as 50% of HCM cases, the genetic cause remains unknown, suggesting that other genes may be involved. METHODS Clinical evaluation, including clinical history, physical examination, electrocardiography, and 2-dimensional echocardiography, was performed, and blood was collected from family members (n = 23) for deoxyribonucleic acid analysis. The family was genotyped with markers from the 10-cM AB PRISM Human Linkage mapping set (Applied Biosystems, Foster City, California), and 2-point linkage analysis was performed. RESULTS Affected family members showed marked clinical diversity, ranging from asymptomatic individuals to those with syncope, heart failure, and premature sudden death. The disease locus for this family was mapped to chromosome 1q42.2-q43, near the marker D1S2850 (logarithm of odds ratio = 2.82, theta = 0). A missense mutation, Ala119Thr, in the alpha-actinin-2 (ACTN2) gene was identified that segregated with disease in the family. An additional 297 HCM probands were screened for mutations in the ACTN2 gene using high-resolution melt analysis. Three causative ACTN2 mutations, Thr495Met, Glu583Ala, and Glu628Gly, were identified in an additional 4 families (total 1.7%) with HCM. CONCLUSIONS This is the first genome-wide linkage analysis that shows mutations in ACTN2 cause HCM. Mutations in genes encoding Z-disk proteins account for a small but significant proportion of genotyped HCM families.

Molecular Mechanism of Energy Conservation in Polysulfide Respiration.

Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-Å resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water-filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, using polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAFWT (C8161 and WM852) and BRAFV600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAFWT and BRAFV600E melanoma.

Protonmotive force generation by a redox loop mechanism

Respiration involves the oxidation and reduction of substrate for the redox‐linked formation of a protonmotive force (PMF) across the inner membrane of mitochondria or the plasma membrane of bacteria. A mechanism for PMF generation was first suggested by Mitchell in his chemiosmotic theory. In the original formulations of the theory, Mitchell envisaged that proton translocation was driven by a ‘redox loop’ between two catalytically distinct enzyme complexes. Experimental data have shown that this redox loop does not operate in mitochondria, but has been confirmed as an important mechanism in bacteria. The nitrate respiratory pathway in Escherichia coli is a paradigm for a protonmotive redox loop. The structure of one of the enzymes in this two‐component system, formate dehydrogenase‐N, has revealed the structural basis for the PMF generation by the redox loop mechanism and this forms the basis of this review.

Potassium-activated GTPase Reaction in the G Protein-coupled Ferrous Iron Transporter B

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe2+). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe2+ membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2′-(or -3′)-O-(N-methylanthraniloyl)-β,γ-imidoguanosine 5′-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K+, which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K+ coordination, and mutation of this residue abolished K+-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K+-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin

In vertebrates, the iron exporter ferroportin releases Fe2+ from cells into plasma, thereby maintaining iron homeostasis. The transport activity of ferroportin is suppressed by the peptide hormone hepcidin, which exhibits upregulated expression in chronic inflammation, causing iron-restrictive anaemia. However, due to the lack of structural information about ferroportin, the mechanisms of its iron transport and hepcidin-mediated regulation remain largely elusive. Here we report the crystal structures of a putative bacterial homologue of ferroportin, BbFPN, in both the outward- and inward-facing states. Despite undetectable sequence similarity, BbFPN adopts the major facilitator superfamily fold. A comparison of the two structures reveals that BbFPN undergoes an intra-domain conformational rearrangement during the transport cycle. We identify a substrate metal-binding site, based on structural and mutational analyses. Furthermore, the BbFPN structures suggest that a predicted hepcidin-binding site of ferroportin is located within its central cavity. Thus, BbFPN may be a valuable structural model for iron homeostasis regulation by ferroportin.

Structural basis of GDP release and gating in G protein coupled Fe2+ transport.

G proteins are key molecular switches in the regulation of membrane protein function and signal transduction. The prokaryotic membrane protein FeoB is involved in G protein coupled Fe2+ transport, and is unique in that the G protein is directly tethered to the membrane domain. Here, we report the structure of the soluble domain of FeoB, including the G protein domain, and its assembly into an unexpected trimer. Comparisons between nucleotide free and liganded structures reveal the closed and open state of a central cytoplasmic pore, respectively. In addition, these data provide the first observation of a conformational switch in the nucleotide‐binding G5 motif, defining the structural basis for GDP release. From these results, structural parallels are drawn to eukaryotic G protein coupled membrane processes.

Purification and crystallization of the respiratory complex formate dehydrogenase-N from Escherichia coli

A membrane-protein complex, formate dehydrogenase-N from Escherichia coli, has been purified and crystallized. This molybdenum-containing enzyme, composed of alpha, beta and gamma subunits, is the major electron donor to the nitrate respiratory chain of E. coli. The formate dehydrogenase-N crystals belong to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 203 A. An asymmetric unit of the crystals is assumed to contain one formate dehydrogenase-N monomer (MW 170 kDa). One data set to 1.6 A resolution, with 342 711 independent observations (94.4% complete) and an R(merge) of 0.08, has been collected from a single crystal. This is the highest resolution data set reported for a membrane-protein complex to date.

The Initiation of GTP Hydrolysis by the G-Domain of FeoB: Insights from a Transition-State Complex Structure

The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoBSt) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF4 −, reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoBSt, and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoBSt. Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.

The cation-dependent G-proteins: In a class of their own

G‐proteins are some of the most important and abundant enzymes, yet their intrinsic nucleotide hydrolysis reaction is notoriously slow and must be accelerated in vivo. Recent experiments on dynamin and GTPases involved in ribosome assembly have demonstrated that their hydrolysis activities are stimulated by potassium ions. This article presents the hypothesis that cation‐mediated activation of G‐proteins is more common than currently realised, and that such GTPases represent a structurally and functionally unique class of G‐proteins. Based on sequence analysis we provide a list of predicted cation‐dependent GTPases, which encompasses almost all members of the TEES, Obg‐HflX, YqeH‐like and dynamin superfamilies. The results from this analysis effectively re‐define the conditions under which many of these G‐proteins should be studied in vitro.