The structural integrity of TDP-43 N-terminus is required for efficient aggregate entrapment and consequent loss of protein function

ABSTRACT. Nuclear factor TDP-43 has been shown to play a key role in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia, where TDP-43 aggregates accumulate in patient's affected neurons and this event can cause neuronal dysfunction. A major focus of today's research is to discover the critical factors that lead to TDP-43 aggregation and the consequences for neuronal metabolism. From a structural point of view, several lines of evidence point toward TDP-43 C-terminus as a key domain able to mediate this process. Regarding this region, we have recently described a novel cellular TDP-43 aggregation model based on 12 tandem repetitions of its 339-366 Q/N rich prion-like domain. In addition, we have shown and confirmed that a minimal TDP-43 construct constituted by the N and C-terminal regions, but lacking both RRM domains, induce aggregation of endogenous TDP-43 and leads to its total loss of function as seen by changes in the alternative splicing of endogenous genes. In this work, we further characterize this model and show the importance of the N-terminus structure in the loss of function process. In addition, from a biochemical point of view we report that, as shown in a previous version of this model (GFP 12×Q/N), the endogenous TDP-43 trapped in the aggregates undergoes the 2 most important post-translational modifications seen in pathological TDP-43 inclusions: ubiquitination and hyperphosphorylation.     

The C-terminal amphipathic α-helix of Pseudomonas aeruginosa PelC outer membrane protein is required for its function

Pseudomonas aeruginosa is an opportunistic pathogen, which causes numerous infections and can adopt a versatile lifestyle. During chronic infection, P. aeruginosa becomes established as a bacterial community known as a biofilm. Biofilm formation results from the production of a matrix mainly comprised of exopolysaccharides. P. aeruginosa possesses several gene clusters which contribute to the formation of the matrix, including the pel genes. Among the pel genes, pelC encodes an outer membrane protein, which may serve as a transporter of polysaccharide to the bacterial cell surface. Whereas outer membrane proteins usually display an amphipathic β-barrel fold, we show that PelC requires a C-terminal amphipathic α-helix for outer membrane insertion and function. Such a structural feature has only previously been reported for the Wza outer membrane protein of Escherichia coli, and our data suggest that this characteristic may be found in a large family of proteins, particularly outer membrane proteins specialized in polysaccharide transport.     

The PprA protein is required for accurate cell division of γ-irradiated Deinococcus radiodurans bacteria

Deinococcus radiodurans, one of the most radioresistant organisms known to date is able to reconstruct an intact genome from hundreds of DNA fragments. Here, we investigate the in vivo role of PprA, a radiation-induced Deinococcus specific protein. We report that DNA double strand break repair in cells devoid of PprA and exposed to 3800 Gy γ-irradiation takes place efficiently with a delay of only 1 h as compared to the wild type, whereas massive DNA synthesis begins 90 min after irradiation as in the wild type, a phenotype insufficient to explain the severe radiosensitivity of the ΔpprA mutant. We show that the slow kinetics of reassembly of DNA fragments in a ΔpprA ΔrecA double mutant was the same as that observed in a ΔrecA single mutant demonstrating that PprA does not play a major role in DNA repair through RecA-independent pathways. Using a tagged PprA protein and immunofluorescence microscopy, we show that PprA is recruited onto the nucleoid after γ-irradiation before DNA double strand break repair completion, and then is found as a thread across the septum in dividing cells. Moreover, whereas untreated cells devoid of PprA displayed a wild type morphology, they showed a characteristic cell division abnormality after irradiation not found in other radiosensitive mutants committed to die, as DNA is present equally in the two daughter cells but not separated at the division septum. We propose that PprA may play a crucial role in the control of DNA segregation and/or cell division after DNA double strand break repair.     

What is the function of protein carboxyl methylation?

The following functions of protein carboxyl methylation seem to be reasonably well established: Multiple, stoichiometric methylation of chemotactic receptors in bacteria at glutamyl residues serves as one (but not the only) adaptation mechanism of the transduction chain to constant background levels of chemotactic stimuli. Stoichiometric methylation of hormones and hormone carrier proteins plays a role in hormone storage and secretion by the pituitary gland. Substoichiometric methylation at D-aspartyl residues is involved in a repair mechanism of aged proteins. Stoichiometric methylation of calmodulin modulates the sensitivity of calmodulin-dependent processes to calcium. Research of the past 3 years has indicated that in order to demonstrate an involvement of methylation in the coupling of surface receptors to intracellular events three new criteria have to be met: (a) the cell should possess a protein carboxyl methylase with relatively narrow substrate specificity; (b) methylation should take place at L-amino acid residues; (c) the methyl accepting proteins should be methylated in a stoichiometric fashion.     

The protein–protein interactions required for assembly of the Tn3 resolution synapse

The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer–dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis (“Bart”). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific “R” interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer–dimer interaction required for catalysis.     

Is loss of function of the prion protein the cause of prion disorders?

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.     

The structural basis of non-photochemical quenching is revealed?

Light-harvesting complex II (LHCII, the major plant light-harvesting pigment-protein complex, efficiently harvests light-energy. However, if the incident light intensity is too high and photosynthesis becomes saturated, LHCII can switch into a quenching state that prevents photodamage. This important process is called non-photochemical quenching, or NPQ, and represents feedback control. Andrew Pascal et al. have recently proposed a detailed model of NPQ based upon the crystal structure of LHCII from spinach.     

The fate of duplicated genes: loss or new function?

Gene duplication events are important sources of novel gene functions. However, more often than not, a duplicate gene may lose its function and become a pseudogene. What is the relative frequency of these two scenarios: functional divergence versus gene loss? Given that most non-neutral mutations are deleterious, gene loss should be far more frequent than divergence. However, a recent empirical study suggests that about 50% of all gene duplications will lead to functional divergence. The study infers the frequency of functional divergence from the size distribution of gene families produced by two successive genome duplications early in vertebrate evolution. Reasons for this unexpectedly high frequency of functional divergence are discussed.     

Adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetic mice

Rosiglitazone is a PPARγ agonist commonly used to treat diabetes. In addition to improving insulin sensitivity, rosiglitazone restores normal vascular function by a mechanism that remains poorly understood. Here we show that adiponectin is required to mediate the PPARγ effect on vascular endothelium of diabetic mice. In db/db and diet-induced obese mice, PPARγ activation by rosiglitazone restores endothelium-dependent relaxation of aortae, whereas diabetic mice lacking adiponectin or treated with an anti-adiponectin antibody do not respond. Rosiglitazone stimulates adiponectin release from fat explants, and subcutaneous fat transplantation from rosiglitazone-treated mice recapitulates vasodilatation in untreated db/db recipients. Mechanistically, adiponectin activates AMPK/eNOS and cAMP/PKA signaling pathways in aortae, which increase NO bioavailability and reduce oxidative stress. Taken together, these results demonstrate that adipocyte-derived adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetes. Thus, the adipose tissue represents a promising target for treating diabetic vasculopathy.     

The carbohydrate-binding domain of overexpressed STBD1 is important for its stability and protein–protein interactions

STBD1 (starch-binding domain-containing protein 1) belongs to the CBM20 (family 20 carbohydrate binding module) group of proteins, and is implicated in glycogen metabolism and autophagy. However, very little is known about its regulation or interacting partners. Here, we show that the CBM20 of STBD1 is crucial for its stability and ability to interact with glycogen-associated proteins. Mutation of a conserved tryptophan residue (W293) in this domain abolished the ability of STBD1 to bind to the carbohydrate amylose. Compared with the WT (wild-type) protein, this mutant exhibited rapid degradation that was rescued upon inhibition of the proteasome. Furthermore, STBD1 undergoes ubiquitination when expressed in COS cells, and requires the N-terminus for this process. In contrast, inhibition of autophagy did not significantly affect protein stability. In overexpression experiments, we discovered that STBD1 interacts with several glycogen-associated proteins, such as GS (glycogen synthase), GDE (glycogen debranching enzyme) and Laforin. Importantly, the W293 mutant of STBD1 was unable to do so, suggesting an additional role for the CBM20 domain in protein–protein interactions. In HepG2 hepatoma cells, overexpressed STBD1 could associate with endogenous GS. This binding increased during glycogenolysis, suggesting that glycogen is not required to bridge this interaction. Taken together, our results have uncovered new insights into the regulation and binding partners of STBD1.     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

What is the minimum number of letters required to fold a protein?

Experimental studies have shown that the full sequence complexity of naturally occurring proteins is not required to generate rapidly folding and functional proteins, i.e. proteins can be designed with fewer than 20 letters. This raises the question of what is the minimum number of amino acid types required to encode complex protein folds? Here, we investigate this issue from three aspects. First, we study the minimum sequence complexity that can reserve the necessary structural information for detection of distantly related homologues. Second, we compare the ability of designing foldable model sequences over a wide range of reduced amino acid alphabets, which find the minimum number of letters that have the similar design ability as 20. Finally, we survey the lower bound of alphabet size of globular proteins in a non-redundant protein database. These different approaches give a remarkably consistent view, that the minimum number of letters required to fold a protein is around ten.     

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem–loop sequence, which is bound to the stem–loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′hExo, forms a ternary complex with SLBP and the stem–loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3′hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3′hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.     

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.     

The GCP3-interacting proteins GIP1 and GIP2 are required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability

Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.     

Transforming growth factor β receptor type 1 is essential for female reproductive tract integrity and function

The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1-mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.