Two Mosquito LRR Proteins Function as Complement Control Factors in the TEP1-Mediated Killing of Plasmodium

SummaryPlasmodium development within Anopheles mosquitoes is a vulnerable step in the parasite transmission cycle, and targeting this step represents a promising strategy for malaria control. The thioester-containing complement-like protein TEP1 and two leucine-rich repeat (LRR) proteins, LRIM1 and APL1, have been identified as major mosquito factors that regulate parasite loads. Here, we show that LRIM1 and APL1 are required for binding of TEP1 to parasites. RNAi silencing of the LRR-encoding genes results in deposition of TEP1 on Anopheles tissues, thereby depleting TEP1 from circulation in the hemolymph and impeding its binding to Plasmodium. LRIM1 and APL1 not only stabilize circulating TEP1, they also stabilize each other prior to their interaction with TEP1. Our results indicate that three major antiparasitic factors in mosquitoes jointly function as a complement-like system in parasite killing, and they reveal a role for LRR proteins as complement control factors

A Novel Cysteine-rich Domain of Sep15 Mediates the Interaction with UDP-glucose:Glycoprotein Glucosyltransferase

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 copurifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoproteinM(SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT

NMR Structures of the Selenoproteins Sep15 and SelM Reveal Redox Activity of a New Thioredoxin-like Family

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxinlike protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1.

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1.

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

The basic helix–loop–helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ∼24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here we show that CRY1 binds directly to the PAS domain core of CLOCK:BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solution X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1

DAP-Type PG-Binding Activities of the S158A/C and H42A Mutants and the Structure of the S158-H42 Dyad in the Active Site

<div><p>(A) Upper panel shows the wild-type and mutant rPGRP-SA pulled down by DAP-type PG from <i>E. coli.</i> Lower panel (Input) shows the corresponding protein samples (20 μg) without incubation with PG.</p> <p>(B) Stereo diagram showing the putative active-site residues. Prepared with Bobscript, GL_RENDER, and POV-Ray.</p></div

PGRP-SA Is an L,D-Carboxypeptidase

<div><p>(A) Chemical structures of the DAP-type muropeptide, GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-<i>meso</i>-<b>DAP</b>-D-Ala, and lysine-type muropeptide, GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-L-<b>Lys</b>-D-Ala, used in the enzymatic assays (substrates S₁ and S₂, respectively). The arrow indicates the site of cleavage of the DAP-type substrate S₁ by the L,D-carboxypeptidase activity.</p> <p>(B–G) Reverse-phase HPLC analysis. Cleavage of the DAP-type substrate S₁ by wild-type rPGRP-SA results in the generation of GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-<i>meso</i>-DAP (P₁ product). The position of the peak corresponding to standard GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-L-Lys (P₂ product, not generated by rPGRP-SA) is indicated.</p> <p>(B) Incubation of S₁ without rPGRP-SA for 40 h.</p> <p>(C and D) Incubation of S₁ with rPGRP-SA for (C) 24 h and (D) 40 h.</p> <p>(E) Incubation of S₂ with rPGRP-SA for 70 h.</p> <p>(F and G) Incubation of S₁ with the (F) S158C and (G) H42A mutants for 40 h.</p></div

PGRP-SA Structure and Sequence Comparisons

<div><p>(A) Ribbon diagram showing the front view (left) and side view (right) of PGRP-SA. The ribbon is colored from N to C terminus in a progression from blue to red. Disulfide bridges are shown as sticks. The π helix turn at the end of the H2 helix is indicated.</p> <p>(B) Comparison of PGRP-SA (blue coil, from N to C) and PGRP-LB (green coil, from N′ to C′). (A) and (B) were prepared with Bobscript (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020277#pbio-0020277-Esnouf1" target="_blank">Esnouf 1999</a>), GL_RENDER (E. Esser, personal communication), and POV-Ray (Persistence of Vision Ray Tracer v3.1g).</p> <p>(C) Aligned sequences of selected PGRP domains, with a serine and a histidine at position 158 and position 42 of PGRP-SA (marked with asterisks), respectively, from <i>Drosophila</i> (d), mouse (m), and human (h). Secondary-structure elements in PGRP-SA are indicated above the alignment. Invariant residues are boxed in black and colored in white, conserved residues are shaded in yellow, and those lining the putative PG docking groove are in pink. The disulfide bond-forming Cys residues are boxed in gray. The residue number of PGRP-SA is shown above the alignment. The residues chosen for mutagenesis are marked with black circles. A structurally based alignment of the dPGRP-LB sequence is shown at the bottom with its amidase catalytic zinc-coordinating residues colored in red.</p></div

Mutational Analysis of PGRP-SA

<div><p>(A) Upper panel shows the wild-type and mutant rPGRP-SA pulled down by lysine-type PG from <i>M. luteus.</i> Lower panel (Input) shows the corresponding protein samples (20 μg) without incubation with PG.</p> <p>(B) The relative Toll signaling activities of the rPGRP-SA mutants. At least three repeats were performed for each experiment. Each bar represents the mean with the standard deviation. The values obtained for the wild type after M. luteus PG injection were arbitrarily set to 100 (upper dashed line). The background activity level is indicated by the lower dashed line. CTR, unchallenged control.</p></div