Capturing cellular machines by systematic screens of protein complexes

Two recent studies have provided the most complete screen for protein complexes in yeast to date, in which partners were identified for approximately half of the proteome. A comparison shows that these two datasets are complementary. In addition, one of the analyses points to a modular organization of the cellular protein network. These data will prove useful in defining principles and trends that arise when combining large-scale datasets of different natures, and in deriving properties of protein machines in cellular systems.     

Recapitulation of the effects of the human papillomavirus type 16 E7 oncogene on mouse epithelium by somatic Rb deletion and detection of pRb-independent effects of E7 in vivo

Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.     

SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.     

Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis

The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-Å crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg³⁷ plays a crucial role in the hydrolysis of the thioester bond, and that Tyr²⁹ and a hydrogen-bonded water network assist the decarboxylation of the β-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4=C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.Enediyne natural products represent a family of structurally unique secondary metabolites with potent antitumor and antibiotic activities. Based on the structure of the bicyclic enediyne core, enediyne natural products are categorized into two groups with either a 9- or 10-membered enediyne moiety (1, 2). The antitumor activity of enediyne natural products derives from their capacity to induce chromosomal DNA cleavage through an oxidative radical mechanism (3). The biosynthetic mechanism for the enediyne moiety has been, however, elusive despite clues gleaned from early isotope-feeding experiments (4, 5). Pioneering genetic studies of the biosynthesis of calicheamicin and C-1027 from two research groups yielded major insights into the biosynthetic pathways, suggesting that an iterative polyketide synthase (PKS)5 plays a central role in the assembly of both the 9- and 10-membered enediyne moieties (6, 7). The gene clusters also contain open reading frames encoding hypothetical proteins for the downstream processing of the PKS product. The involvement of similar genes in enediyne biosynthesis was later confirmed for neocarzinostatin, maduropeptin, dynemicin, and several putative enediyne natural products in soil and marine microorganisms (8–11). Recently, based on the study on the 9-membered enediyne-containing C-1027, Shen and coworkers found that the iterative PKS (SgcE) and the putative thioesterase (SgcE10) generated a conjugated polyene (1,3,5,7,9,11,13-pentadecaheptaene) through an ACP-tethered 3-hydroxy-4,6,8,10,12,14-hexadecahexaene intermediate during co-expression in Escherichia coli (12). The release of the product catalyzed by the putative thioesterase SgcE10 presumably occurs through a combination of hydrolysis, decarboxylation, and dehydration steps. Recent biochemical studies of the iterative PKS (CalE8) from the biosynthetic pathway of calicheamicin also provided insight into the early steps of 10-membered enediyne biosynthesis (13, 14). It was observed that CalE8 produced a linear carbonyl-conjugated polyene (3,5,7,9,11,13-pentadecen-2-one (1)) with the assistance of the putative thioesterase CalE7 (Fig. 1). The putative biosynthetic intermediate 1 was proposed to derive from a 16-carbon-long β-ketocarboxylic intermediate tethered to CalE8 (13). Given the loss of one carbon unit during product release, a decarboxylation process was speculated to occur following the hydrolysis of the thioester bond.Open in a separate windowFIGURE 1.Calicheamicin and its biosynthesis. A, structure of calicheamicin γ′₁ with the incorporated acetate units in the 10-membered enediyne moiety highlighted in bold sticks. B, early steps of the biosynthetic pathway of the 10-membered enediyne as proposed by Kong et al. (13). The incorporated acetate units are highlighted in bold sticks with the configuration of the double bonds in the intermediates arbitrarily assigned. (AT, acyl transferase; KS, ketoacyl synthase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; and PPTase, phosphopantetheinyl transferase.).Polyketide and non-ribosomal peptide synthesis generally involves the so-called type I and type II thioesterases for the release of final product or removal of aberrant products. Type I thioesterases (TE I) are cis-acting domains fused to the C terminus of the most downstream module of PKS or non-ribosomal peptide synthase for the release and cyclization of the final product (15, 16). By contrast, type II thioesterases (TE II) are discrete proteins responsible for the trans hydrolytic release of aberrant products (17–19). TE II proteins are structurally and evolutionarily related to a family of well known α/β hydrolase that contain 240–260 residues (20). A common serine esterase motif GXSXG and another downstream motif GXH are conserved in TE II proteins (21, 22). The stand-alone 146-amino acid-containing CalE7 does not belong to the TE II family, because it is neither an α/β fold hydrolase nor a protein containing the two conserved motifs for TE II. Instead, CalE7 shares moderate sequence homology with a family of hotdog fold proteins characterized by a long central α-helix packed against a five-stranded anti-parallel β-sheet. Such hotdog fold proteins include many characterized and hypothetical thioesterases that use acyl CoA as substrates (23). The three-dimensional structure and substrate specificity of several hotdog fold thioesterases have been determined, including YbgC from Helicobacter pylori (24), Paal from E. coli (25), HB8 from Thermos thermophilis (26), FcoT from Mycobacterium tuberculosis (27), YciA from Haemophilus influenzae (28), human THEM2 (25) and 4-hydroxylbenzoyl-CoA thioesterases (4-HBT) from Pseudomonas sp. Strain CBS and Arthrobacter sp. strain SU (29–31). Despite their diverse specificity toward acyl substrates (23, 25), all known hotdog fold thioesterases catalyze the hydrolysis of thioester bond using a Glu/Asp residue as nucleophile or general-base catalyst with the exception of FcoT (27). Here we present structural and biochemical data showing that CalE7 does not contain an acidic residue in its active site and is thus likely to utilize a different catalytic mechanism. The results also suggest that CalE7 facilitates a subsequent decarboxylation step to yield the carbonyl-conjugated polyene (1). Hence, the results introduce a hotdog fold thioesterase with a novel product-releasing mechanism in comparison with the traditional type I and II thioesterases associated with the biosynthesis of polyketide natural products. Furthermore, the crystal structure revealed a kinked substrate-binding channel that is predicted to bind a cis-double bond-containing polyene substrate, raising the possibility that CalE8 is able to generate a cis-double bond.     

Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death

Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity.     

Climate-driven spatial and temporal patterns in peatland pool biogeochemistry

Peatland pools are freshwater bodies that are highly dynamic aquatic ecosystems because of their small size and their development in organic-rich sediments. However, our ability to understand and predict their contribution to both local and global biogeochemical cycles under rapidly occurring environmental change is limited because the spatiotemporal drivers of their biogeochemical patterns and processes are poorly understood. We used (1) pool biogeochemical data from 20 peatlands in eastern Canada, the United Kingdom, and southern Patagonia and (2) multi-year data from an undisturbed peatland of eastern Canada, to determine how climate and terrain features drive the production, delivering and processing of carbon (C), nitrogen (N), and phosphorus (P) in peatland pools. Across sites, climate (24%) and terrain (13%) explained distinct portions of the variation in pool biogeochemistry, with climate driving spatial differences in pool dissolved organic C (DOC) concentration and aromaticity. Within the multi-year dataset, DOC, carbon dioxide (CO₂), total N concentrations, and DOC aromaticity were highest in the shallowest pools and at the end of the growing seasons, and increased gradually from 2016 to 2021 in relation to a combination of increases in summer precipitation, mean air temperature for the previous fall, and number of extreme summer heat days. Given the contrasting effects of terrain and climate, broad-scale terrain characteristics may offer a baseline for the prediction of small-scale pool biogeochemistry, while broad-scale climate gradients and relatively small year-to-year variations in local climate induce a noticeable response in pool biogeochemistry. These findings emphasize the reactivity of peatland pools to both local and global environmental change and highlight their potential to act as widely distributed climate sentinels within historically relatively stable peatland ecosystems.     

Androgenesis is a maternal trait in the invasive ant Wasmannia auropunctata

Androgenesis is the production of an offspring containing exclusively the nuclear genome of the fathering male via the maternal eggs. This unusual mating system is generally considered a male trait, giving to androgenetic males a substantial fitness advantage over their sexually reproducing relatives. We here provide the first empirical study of the evolutionary outcomes of androgenesis in a haplo-diploid organism: the invasive ant Wasmannia auropunctata. Some of the populations of this species have a classical haplo-diploid sexual mating system. In other populations, females and males are produced through parthenogenesis and androgenesis, respectively, whereas workers are produced sexually. We conducted laboratory reciprocal-cross experiments with reproductive individuals from both types of populations and analysed their progenies with genetic markers, to determine the respective contribution of males and females to the production of androgenetic males. We found that androgenesis was a parthenogenetic female trait. A population genetic study conducted in natura confirmed the parthenogenetic female origin of androgenesis, with the identification of introgression events of sexual male genotypes into androgenetic/parthenogenetic lineages. We argue that by producing males via androgenesis, parthenogenetic queen lineages may increase and/or maintain their adaptive potential, while maintaining the integrity of their own genome, by occasionally acquiring new male genetic material and avoiding inbreeding depression within the sexually produced worker cast.     

A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults

Background

Strategies to enhance the immunogenicity of DNA vaccines in humans include i) co-administration of molecular adjuvants, ii) intramuscular administration followed by in vivo electroporation (IM/EP) and/or iii) boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study.

Methods

Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG) plasmid DNA (pDNA) vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12) (GENEVAX IL-12) given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM.

Results

All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs) were reported. T cell and antibody response rates after HIVMAG (x3) prime—Ad35 (x1) boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ) ELISPOT responses was highest after HIVMAG (x3) without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS) were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3) prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected.

Conclusion

The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected.

Trial Registration

ClinicalTrials.gov NCT01496989     

Kinetic effects of fiber type on the two subcomponents of the Huxley-Simmons phase 2 in muscle

The Huxley-Simmons phase 2 controls the kinetics of the first stages of tension recovery after a step-change in fiber length and is considered intimately associated with tension generation. It had been shown that phase 2 is comprised of two distinct unrelated phases. This is confirmed here by showing that the properties of phase 2(fast) are independent of fiber type, whereas those of phase 2(slow) are fiber type dependent. Phase 2(fast) has a rate of 1000-2000 s(-1) and is temperature insensitive (Q(10) approximately 1.16) in fast, medium, and slow speed fibers. Regardless of fiber type and temperature, the amplitude of phase 2(fast) is half (approximately 0.46) that of phase 1 (fiber instantaneous stiffness). Consequently, fiber compliance (cross-bridge and thick/thin filament) appears to be the common source of both phase 1 elasticity and phase 2(fast) viscoelasticity. In fast fibers, stiffness increases in direct proportion to tension from an extrapolated positive origin at zero tension. The simplest explanation is that tension generation can be approximated by two-state transition from attached preforce generating (moderate stiffness) to attached force generating (high stiffness) states. Phase 2(slow) is quite different, progressively slowing in concert with fiber type. An interesting interpretation of the amplitude and rate data is that reverse coupling of phase 2(slow) back to P(i) release and ATP hydrolysis appears absent in fast fibers, detectable in medium speed fibers, and marked in slow fibers contracting isometrically. Contracting slow and heart muscles stretched under load could employ this enhanced reversibility of the cross-bridge cycle as a mechanism to conserve energy.