The evolving doublecortin (DCX) superfamily

BACKGROUND: Doublecortin (DCX) domains serve as protein-interaction platforms. Mutations in members of this protein superfamily are linked to several genetic diseases. Mutations in the human DCX gene result in abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, and DCDC2 has been associated with dyslectic reading disabilities. RESULTS: The DCX-repeat gene family is composed of eleven paralogs in human and in mouse. Its evolution was followed across vertebrates, invertebrates, and was traced to unicellular organisms, thus enabling following evolutionary additions and losses of genes or domains. The N-terminal and C-terminal DCX domains have undergone sub-specialization and divergence. Developmental in situ hybridization data for nine genes was generated. In addition, a novel co-expression analysis for most human and mouse DCX superfamily-genes was performed using high-throughput expression data extracted from Unigene. We performed an in-depth study of a complete gene superfamily using several complimentary methods. CONCLUSION: This study reveals the existence and conservation of multiple members of the DCX superfamily in different species. Sequence analysis combined with expression analysis is likely to be a useful tool to predict correlations between human disease and mouse models. The sub-specialization of some members due to restricted expression patterns and sequence divergence may explain the successful addition of genes to this family throughout evolution

Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels

The upper interface pivotal T248 residue affects channel maturation and gating.

<p><b>A</b>) K⁺ currents recorded from <i>Xenopus laevis</i> oocytes expressing the wild type channel, the T248A and L249T single-mutants and the corresponding T248A;L249T double-mutant. <b>B</b>) Voltage-activation data for the K⁺ channel proteins indicated in (<b>A</b>). In all panels, smooth curves correspond to a two-state Boltzmann function. For clarity, in this and in other figures, no error bars are plotted for the different data points of the different curves. These values, however, are in a range of 5% of the reported values.</p

The upper interface T248, Y415 and S428 residue triad forms a network of coupled interactions during activation gating.

<p>A) A thermodynamic cubic construct was used to measure mutual three-dimensional coupling (<i>Δ</i>³<i>G_(i,j,k)</i>) between the three upper interface residues. <i>Δ</i>³<i>G</i> is calculated by subtracting the coupling free energy between any residue pair in the presence and absence of the native third residue (front and back faces of the cube, respectively). For clarity, only the channel proteins of the back face are explicitly represented. The front face proteins are described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone-0082253-g004" target="_blank">Fig. 4A</a>. B) Voltage-activation curves for four channel proteins comprising the thermodynamic double-mutant cycle measuring the coupling free energy between the upper interface T248 and Y415 (<i>i,j</i>) residues on the background of the mutated S428 residue (<i>k</i>). The same cycle, only in the presence of the native third position, is considered in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone-0082253-g004" target="_blank">Fig. 4B</a> (front face of the cubic construct shown in (A)).</p

The upper interface single-mutant gating parameters conform to the expected trend for perturbations that primarily affect late concerted pore opening.

<p>The Z and V_1/2 phenomenological gating parameter values of the T248A, Y415A, S428 and I429A upper interface single-mutants mapped onto the Z-V_1/2 phase space (red circles), in the context of a previously observed experimental correlation between the Z and V_1/2 values of pore mutant <i>Shaker</i> channel proteins (black circles). The green point is for the wild type <i>Shaker</i> channel values. The smooth curve corresponds to the expected theoretical trend for the 16-state gating model of the <i>Shaker</i> channel (Zagotta, Hoshi and Aldrich (1994), <i>J Gen Physiol</i> 103: 321-362), where perturbations are assumed to only affect late concerted pore opening (<i>L</i>) transition. Refer to reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone.0082253-Yifrach1" target="_blank">[11]</a> for further details.</p

The influence of upper interface single-, double- and triple-mutations on slow inactivation gating of the tandem dimer <i>Shaker</i> Kv channel^a

<p><i>K</i>_I) and the free energy for inactivation gate closure (<i>ΔG</i>_inactivation), for all single-, double- and triple-mutants used to calculate the coupling free energies during inactivation gating.^a The table displays the forward and backward time constants for slow inactivation gating (at 0 mV) along with the inactivation equilibrium constant (</p><p><i>K</i>_I were calculated according to <i>K</i>_I = <i>k</i>_f/<i>k</i>_r (see <b>Materials and Methods</b>).^b Values for </p><p><i>ΔG</i>_inactivation were calculated according to <i>ΔG</i>_inactivation = −RTln <i>K</i>_I.^c Values for </p><p><b>Materials and Methods</b>).^d Values for the slow recovery phase are presented (see </p><p><i>ΔΔG</i>_inactivation were calculated according to <i>ΔΔG</i>_inactivation = (−<i>RT</i>ln (<i>k</i>_f/<i>k</i>_b)_wt −−<i>RT</i>ln (<i>k</i>_f/<i>k</i>_b)_m).^e Values for </p

Residues across the upper interface are thermodynamically coupled in a state-dependent manner.

<p><b>A</b>) Thermodynamic double-mutant cycle coupling analysis of voltage-dependent gating applied to inter-subunit interactions across the upper interface. The equilibrium between the closed and open states of the tandem-dimer wild type (A_WTB_WT), single-mutant (A_M1B_WT or A_WTB_M2), and double-mutant (A_M1B_M2) channels is related by a thermodynamic square that enables measurement of the magnitude (<i>Δ</i>²<i>G_(i,j)</i>) and state-dependency (sign of coupling, whether ‘+’ or ‘−’) of pairwise coupling between residues <i>i</i> and <i>j</i> (positions 1 and 2) of the identical A and B subunits, respectively. In the thermodynamic cycle shown, the interaction between the Y415 and T248 residue pair is considered. Mutations (to alanine) are represented by the black color. <b>B–E</b>), Voltage-activation curves for the four channel proteins comprising the double-mutant cycle measuring the coupling free energy between the upper interface T248 and Y415 (<b>B</b>), T248 and S428 (<b>C</b>), T248 and I429 (<b>D</b>) and Y415 and S428 (<b>E</b>) residue pairs. Smooth curves correspond to a two-state Boltzmann function. The smooth gray curve in each panel corresponds to an extrapolated voltage-activation curve that assumes additivity of the effects of the two single-mutations on gating (see <b>Materials and Methods</b>).</p

The intimate upper interface residue network loosens upon Kv channel closure.

<p>Side and top view comparisons of the upper interface residue network between the open Kv channel structure (left) and the different models of the closed channel state (indicated by the reference notation above each rectangle). Structures were first aligned using the sPDB viewer to allow a common coordinate system. Panel figures were prepared using the Molecular graphics UCSF Chimera package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone.0082253-Pettersen1" target="_blank">[34]</a>. The number in each panel represents the shortest atom distance (in Å) between the T248 and Y415 residue pair. Note that in two closed state models (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone.0082253-Jensen1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone.0082253-KhaliliAraghi1" target="_blank">[33]</a>), apparent asymmetry is observed along the upper domain interface. Space-filling representations of the Kv S1 T248, S5 Y415 and P S428 residues are highlighted in blue, gold and green, respectively, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082253#pone-0082253-g001" target="_blank"><b>Fig 1C</b></a>.</p