Inflammation and repeated infections in CGD: two sides of a coin

Chronic granulomatous disease (CGD) is an uncommon congenital immunodeficiency seen approximately in 1 of 250,000 individuals. It is caused by a profound defect in a burst of oxygen consumption that normally accompanies phagocytosis in all myeloid cells (neutrophils, eosinophils, monocytes, and macrophages). This “respiratory burst” involves the catalytic conversion of molecular oxygen to the oxygen free-radical superoxide, which in turn gives rise to hydrogen peroxide, hypochlorous acid, and hydroxyl radicals. These oxygen derivatives play a critical role in the killing of pathogenic bacteria and fungi. As a result of the failure to activate the respiratory burst in their phagocytes, the majority of CGD patients suffer from severe recurrent infections and rather unexplained prolonged inflammatory reactions that may result in granulomatous lesions. Both may cause severe organ dysfunction depending on the tissues involved. Preventive measures as well as rapid (invasive) diagnostic procedures are required to successfully treat CGD. Hematopoietic stem cell transplantation may be a serious option in some of the patients.     

Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.     

Key role of integrin αIIbβ3 signaling to Syk kinase in tissue factor-induced thrombin generation

The fibrin(ogen) receptor, integrin α(IIb)β(3), has a well-established role in platelet spreading, aggregation and clot retraction. How α(IIb)β(3) contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used α(IIb)β(3) blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca(2+) responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in α(IIb)β(3) signaling similarly enhances thrombin-induced Ca(2+) rises and PS exposure. These responses are reduced in α(IIb)β(3)-deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of α(IIb)β(3) to tissue factor-induced platelet Ca(2+) rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on α(IIb)β(3) activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca(2+) signal generation, and furthermore that a considerable part of Syk activation relies on α(IIb)β(3) signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation.     

Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex

The loading of antigenic peptides onto major histocompatibility complex class I (MHC I) molecules is an essential step in the adaptive immune response against virally or malignantly transformed cells. The ER-resident peptide-loading complex (PLC) consists of the transporter associated with antigen processing (TAP1 and TAP2), assembled with the auxiliary factors tapasin and MHC I. Here, we demonstrated that the N-terminal extension of each TAP subunit represents an autonomous domain, named TMD(0), which is correctly targeted to and inserted into the ER membrane. In the absence of coreTAP, each TMD(0) recruits tapasin in a 1:1 stoichiometry. Although the TMD(0)s lack known ER retention/retrieval signals, they are localized to the ER membrane even in tapasin-deficient cells. We conclude that the TMD(0)s of TAP form autonomous interaction hubs linking antigen translocation into the ER with peptide loading onto MHC I, hence ensuring a major function in the integrity of the antigen-processing machinery.     

Nanoparticle-based drug delivery: case studies for cancer and cardiovascular applications

Nanoparticles (NPs) comprised of nanoengineered complexes are providing new opportunities for enabling targeted delivery of a range of therapeutics and combinations. A range of functionalities can be included within a nanoparticle complex, including surface chemistry that allows attachment of cell-specific ligands for targeted delivery, surface coatings to increase circulation times for enhanced bioavailability, specific materials on the surface or in the nanoparticle core that enable storage of a therapeutic cargo until the target site is reached, and materials sensitive to local or remote actuation cues that allow controlled delivery of therapeutics to the target cells. However, despite the potential benefits of NPs as smart drug delivery and diagnostic systems, much research is still required to evaluate potential toxicity issues related to the chemical properties of NP materials, as well as their size and shape. The need to validate each NP for safety and efficacy with each therapeutic compound or combination of therapeutics is an enormous challenge, which forces industry to focus mainly on those nanoparticle materials where data on safety and efficacy already exists, i.e., predominantly polymer NPs. However, the enhanced functionality affordable by inclusion of metallic materials as part of nanoengineered particles provides a wealth of new opportunity for innovation and new, more effective, and safer therapeutics for applications such as cancer and cardiovascular diseases, which require selective targeting of the therapeutic to maximize effectiveness while avoiding adverse effects on non-target tissues.     

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous γH2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the α-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-α primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.     

PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans

Ichthyoses comprise a heterogeneous group of genodermatoses characterized by abnormal desquamation over the whole body, for which the genetic causes of several human forms remain unknown. We used a spontaneous dog model in the golden retriever breed, which is affected by a lamellar ichthyosis resembling human autosomal recessive congenital ichthyoses (ARCI), to carry out a genome-wide association study. We identified a homozygous insertion-deletion (indel) mutation in PNPLA1 that leads to a premature stop codon in all affected golden retriever dogs. We subsequently found one missense and one nonsense mutation in the catalytic domain of human PNPLA1 in six individuals with ARCI from two families. Further experiments highlighted the importance of PNPLA1 in the formation of the epidermal lipid barrier. This study identifies a new gene involved in human ichthyoses and provides insights into the localization and function of this yet uncharacterized member of the PNPLA protein family.     

Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event

Streptococcus pneumoniae ('pneumococcus') causes an estimated 14.5 million cases of serious disease and 826,000 deaths annually in children under 5 years of age(1). The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States(2,3) provided an unprecedented opportunity to investigate the response of an important pathogen to widespread, vaccine-induced selective pressure. Here, we use array-based sequencing of 62 isolates from a US national monitoring program to study five independent instances of vaccine escape recombination(4), showing the simultaneous transfer of multiple and often large (up to at least 44 kb) DNA fragments. We show that one such new strain quickly became established, spreading from east to west across the United States. These observations clarify the roles of recombination and selection in the population genomics of pneumococcus and provide proof of principle of the considerable value of combining genomic and epidemiological information in the surveillance and enhanced understanding of infectious diseases.     

Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas

To identify somatic mutations in pediatric diffuse intrinsic pontine glioma (DIPG), we performed whole-genome sequencing of DNA from seven DIPGs and matched germline tissue and targeted sequencing of an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). We found that 78% of DIPGs and 22% of non-BS-PGs contained a mutation in H3F3A, encoding histone H3.3, or in the related HIST1H3B, encoding histone H3.1, that caused a p.Lys27Met amino acid substitution in each protein. An additional 14% of non-BS-PGs had somatic mutations in H3F3A causing a p.Gly34Arg alteration.     

Maize HapMap2 identifies extant variation from a genome in flux

Whereas breeders have exploited diversity in maize for yield improvements, there has been limited progress in using beneficial alleles in undomesticated varieties. Characterizing standing variation in this complex genome has been challenging, with only a small fraction of it described to date. Using a population genetics scoring model, we identified 55 million SNPs in 103 lines across pre-domestication and domesticated Zea mays varieties, including a representative from the sister genus Tripsacum. We find that structural variations are pervasive in the Z. mays genome and are enriched at loci associated with important traits. By investigating the drivers of genome size variation, we find that the larger Tripsacum genome can be explained by transposable element abundance rather than an allopolyploid origin. In contrast, intraspecies genome size variation seems to be controlled by chromosomal knob content. There is tremendous overlap in key gene content in maize and Tripsacum, suggesting that adaptations from Tripsacum (for example, perennialism and frost and drought tolerance) can likely be integrated into maize.