Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.     

Estrogen actions on follicle formation and early follicle development

Estradiol-17beta (E(2)) affects late follicular development, whereas primordial follicle differentiation and early activation are believed to be independent of E(2). To test this hypothesis we compared numbers of primordial and primary follicles in wild-type and E(2)-deficient, aromatase knockout (ArKO) mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumor 1 (WT-1), and growth differentiation factor (GDF9), which are known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E(2) replacement for 3 wk in 7-wk-old ArKO and wild-type mice on these parameters were also tested. ArKO mice had reduced numbers of primordial and primary follicles compared with wild-type mice (63%, P < 0.001 and 60%, P = 0.062, respectively). This reduction was not corrected by E(2) treatment, suggesting that E(2) affects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared with mice of the wild type. There were no differences in the immunostaining of MIS, WT-1, and PCNA in primordial and primary follicles between wild-type and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells that develop in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E(2) treatment restored the ovarian follicular morphology in ArKO mice, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E(2) has a role in controlling the size of the oocyte and primordial follicle pool in mice.     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

Target antigens determine graft-versus-host disease phenotype

Chronic graft-vs-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Phenotypically, cGVHD differs from patient to patient; in particular, a subset of patients develops extensive cutaneous fibrosis. Similarly, graft-vs-host disease (GVHD) is distinct in inbred murine donor:recipient pairings, indicating a genetic component to disease phenotype. The B10.D2 -->BALB/c (H-2d) strain pairing uniquely recapitulates key pathologic features of fibrotic human cutaneous cGVHD. To distinguish whether this genetic component is due to differences in genes that modulate immune responses or to the specific Ags targeted, we asked whether skin-dominant cGVHD also develops in the B10 -->BALB.B (H-2b) and B10.BR -->BALB.K (H-2k) MHC-congenic pairings. Because each MHC haplotype presents different peptides and selects different T cell repertoires, GVHD in each donor:recipient pair undoubtedly targets different Ags. We found that, in contrast to BALB/c recipients, BALB.B mice never manifested skin disease while BALB.K mice developed a modified form of skin disease. Instead, BALB.B and BALB.K recipients developed systemic GVHD which was absent in BALB/c mice. Moreover, in (B10 x B10.D2)F1 -->(BALB.B x BALB/c)F1 H-2b/d transplants, recipients developed both cutaneous and systemic disease. Thus, the selection of immunodominant Ags determines the target and character of GVHD, providing insight into the genetic basis for different forms of GVHD.     

Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.Human cytomegalovirus (HCMV) (human herpesvirus 5 [HHV-5]), the prototypical betaherpesvirus, is ubiquitous in humans and establishes a persistent infection in the host (19). HCMV also reinfects healthy seropositive individuals, suggesting another mechanism for maintaining persistence in a population (9). Intrauterine transmission and HCMV infection of the developing fetus constitute a leading viral cause of birth defects (32). HCMV is also a leading cause of opportunistic infections in immunocompromised patients, including transplant recipients and patients with AIDS (10, 20). HCMV infection has also been implicated as a cofactor in such diverse diseases as atherosclerosis and cancer (8, 17, 33, 66).HCMV replicates its genome in the nucleus, and acquisition of the final tegument and envelope is thought to occur in the cytoplasm of infected cells (73, 77). Envelopment of HCMV has been reported to occur by budding into cytoplasmic vacuoles that are composed of HCMV glycoproteins required for the assembly of infectious virions (37). The fully mature virus is released from the cell through either exocytosis or, possibly, lysis of the infected cells (56). The nucleic acid-containing capsid is embedded in a proteinaceous tegument layer that occupies the space between the nucleocapsid and the envelope. The tegument contains approximately 40% of the virion protein mass and approximately 20 to 25 known virion proteins, most of which are phosphorylated (40, 44). The assembly pathway and protein interactions required for formation of the tegument layer and the role of individual tegument proteins in the replication and assembly of infectious HCMV remain poorly understood. Deletion of viral genes encoding some tegument proteins results in varying levels of impairment in virus production (11-13, 35, 43, 45, 53, 68). Some tegument proteins, such as pp28 (pUL99) and ppUL25, are expressed only in the cytoplasm of infected cells during HCMV replication, whereas others, such as ppUL53 and pp65 (pUL83), are expressed in the nuclei of cells early in infection but are localized predominantly in the cytoplasm late in infection (68). Others, such as the tegument protein ppUL69, are expressed only in the nuclei of infected cells. Finally, the intracellular localization of other tegument proteins, such as pp150 (pUL32), is less well defined in that both nuclear and cytoplasmic localizations have been described (34, 68).HCMV pp150 (basic phosphoprotein [BPP], pUL32) is the 1,048-amino-acid product of the UL32 gene of HCMV and an abundant constituent of the HCMV virion. Homologues of pp150 are found in other betaherpesviruses, including chimpanzee CMV, rat CMV, mouse CMV, HHV-6, and HHV-7, but not in alpha- or gammaherpesviruses (2). It is expressed late in HCMV infection (15, 68). It comprises 9.1% of infectious virion mass and 2% of the mass of dense bodies, suggesting that it is preferentially incorporated into virions (87). It has an estimated molecular mass of 113 kDa and is posttranslationally modified by phosphorylation and glycosylation, resulting in a molecular mass of 150 kDa in purified virus preparations analyzed by SDS-PAGE (41, 42, 65). pp150 has been classified as a tegument protein based on its presence in virion preparation, noninfectious enveloped particles, and cytoplasmic nucleocapsids but not in immature nuclear capsids (27, 28, 40). It has been suggested that pp150 contacts the capsids through the distal end of the capsomeres or through the triplex subunits that interlink them (16, 86). It has been reported to bind HCMV capsids in vitro through its amino one-third (6). We have also noted association of pp150 with the virion capsid by cryo-immunoelectron microscopy (W. Britt and H. Zhou, UCLA, Los Angeles, CA, unpublished findings). In primary human foreskin fibroblast (HFF) cells infected with HCMV, pp150 accumulates in a juxtanuclear structure that is termed the assembly compartment (AC), which colocalizes with markers of the distal secretory pathway and with other tegument proteins, including pp28 and pp65 and envelope glycoproteins gB, gH, and gM/gN (68). The virus-induced AC appears to overlap with microtubules emanating from the microtubule-organizing center (MTOC) and is proposed to be a cytoplasmic site of virion assembly (37, 68).The function of pp150 is unknown, although its close association with the nucleocapsid suggests potential involvement in nuclear targeting during entry and in nuclear targeting of the encapsidated viral DNA, capsid tegumentation, and/or envelopment late in infection. It is essential for production of infectious virus, since the deletion of the UL32 open reading frame (ORF) leads to loss of virus replication and has been reported to be important in cytoplasmic maturation of HCMV, especially in viral egress (2, 22, 84, 91, 92). In cells infected with ΔUL32 virus, which lacks pp150, fewer virus particles accumulated in the cytoplasm, although nuclear steps in virus assembly were not affected (84). It was also observed that in the absence of pp150, nucleocapsids were present in the viral assembly compartment but failed to proceed further to vesicle transport-associated release (84). These observations, together with pp150 abundance in the virion, suggest a primary contribution for this structural protein in the morphogenesis and/or cytoplasmic transport of progeny virion particles to sites of virion envelopment.Since pp150 has no predicted intracellular trafficking signals, its localization to the AC in virus-infected cells has been postulated to be dependent on interactions with cellular and/or viral proteins. Using yeast two-hybrid (Y2H) screening experiments we identified the cellular protein Bicaudal D1 (BicD1) as an interacting cellular protein. Bicaudal D was originally defined as a Drosophila protein that is involved in establishing the asymmetric cytoplasm in the developing oocyte (82, 89). Two homologues of Bicaudal D, BicD1 and BicD2, have been reported in humans, and these proteins have been reported to be involved in dynein-mediated microtubule transport as well as in COPI-independent Golgi-endoplasmic reticulum (ER) transport (38, 39, 55). Microtubule-dependent transport is an energy-dependent active transport system that includes both positive-end (directed away from the MTOC) and negative-end (directed toward the MTOC) transport. The direction of transport depends on cargo interactions with the molecular motors directing this transport, with dynein being associated with negative-end transport and kinesin with positive-end transport. BicD1 colocalizes with Rab6a in the trans-Golgi network and on cytoplasmic vesicles that associate with Golgi membranes in a Rab6-dependent manner secondary to a Rab6 binding domain at the C terminus of BicD1, suggesting an important role for BicD1 as an adaptor for dynein-dependent transport in the cell (55). In addition to having a role in the Golgi-ER trafficking, BicD1 has been shown to regulate anchoring of microtubules to the centrosome, as BICD1/2 knockdown induced microtubule unfocusing, with microtubules no longer appearing to radiate from the centrosome (26). BicD1 binds to its cargo via its C-terminal domain and to the dynein motor via its N-terminal domain (38). In this study we demonstrated that pp150 and BicD1 interact and that this interaction was required for localization of pp150 to the AC in virus-infected cells. In addition, we demonstrated that inhibition of BicD1 expression by short hairpin RNA (shRNA) led to a reduction in the yield of infectious virus. Finally, we demonstrated that formation of the AC and the assembly of infectious virions were dynein dependent, suggesting a critical role in microtubules in the production of infectious HCMV. Together, these results argue that HCMV replication is dependent on efficient localization of pp150 to the AC through its interaction with BicD1 and that pp150 localization to the AC is dynein dependent.     

Keeping inhibition timely

GABAergic interneurons play a key role in orchestrating cortical network oscillations. In this issue of Neuron, two studies (Bacci and Huguenard and Vida et al.) identify how networks of fast-spiking interneurons can enhance the regularity, precision, and robustness of their own rhythmicity via individual and collective self-innervation.