Search

Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-RING E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo, we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila, rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Ulnar mammary syndrome (UMS) results from heterozygous variants in the TBX3 gene and impacts limb, tooth, hair, apocrine gland, and genitalia development. The expressivity of UMS is highly variable with no established genotype-phenotype correlations. TBX3 belongs to the Tbx gene family, which encodes transcription factors characterized by the presence of a T-box DNA-binding domain. We describe a fetus exhibiting severe upper limb defects and harboring the novel TBX3:c.400 C > T (p.P134S) variant inherited from the mother who remained clinically misdiagnosed until prenatal diagnosis. Literature revision was conducted to uncover the TBX3 clinical and mutational spectrum. Moreover, we generated a Drosophila humanized model for TBX3 to study the developmental consequences of the p.P134S as well as of other variants targeting different regions of the protein. Phenotypic analysis in flies, coupled with in silico modeling on the TBX3 variants, suggested that the c.400 C > T is UMS-causing and impacts TBX3 localization. Comparative analyses of the fly phenotypes caused by the expression of all variants, demonstrated that missense changes in the T-box domain affect more significantly TBX3 activity than variants outside this domain. To improve the clinicians' recognition of UMS, we estimated the frequency of the main clinical features of the disease. Core features often present pre-pubertally include defects of the ulna and/or of ulnar ray, hypoplastic nipples and/or areolas and, less frequently, genitalia anomalies in young males. These results enhance our understanding of the molecular basis and the clinical spectrum of UMS, shedding light on the functional consequences of TBX3 variants in a developmental context., (© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72 , the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. We previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72 -ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. We demonstrate that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72 -related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: ALS is an incurable neuromuscular degenerative disorder. A familiar form of the disease (fALS) is related to point mutations. The most common one is an expansion of a noncoding GGGGCC hexanucleotide repeat of the C9orf72 gene on chromosome 9p21. An abnormal translation of the C9orf72 gene generates dipeptide repeat proteins that aggregate in the brain. One of the classical approaches for developing treatment against protein aggregation-related diseases is to use chemical chaperones (CSs). In this work, we describe the development of novel 4-phenylbutyric acid (4-PBA) lysosome/ER-targeted derivatives. We assumed that 4-PBA targeting to specific organelles, where protein degradation takes place, might reduce the 4-PBA effective concentration., Methods: Organic chemistry synthetic methods and solid-phase peptide synthesis (SPPS) were used for preparing the 4-PBA derivatives. The obtained compounds were evaluated in an ALS Drosophila model that expressed C9orf72 repeat expansion, causing eye degeneration. Targeting to lysosome was validated by the 19 F-nuclear magnetic resonance (NMR) technique., Results: Several synthesized compounds exhibited a significant biological effect by ameliorating the eye degeneration. They blocked the neurodegeneration of fly retina at different efficacy levels. The most active CS was compound 9, which is a peptide derivative and was targeted to ER. Another active compound targeted to lysosome was compound 4., Conclusions: Novel CSs were more effective than 4-PBA; therefore, they might be used as a new class of drug candidates to treat ALS and other protein misfolding disorders.

TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, we demonstrate that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, we show that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, we observe both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-β1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-β1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-β1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten β-tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as "circumferential skin creases Kunze type" (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients' derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients' fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its unfavorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2-isopropyl-4-phenylbutanoic acid (5) has been found to be 2-10-fold more effective than 4-PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes., (© 2018 Wiley Periodicals, Inc.)

Several of the identified genetic factors in Amyotrophic Lateral Sclerosis (ALS) point to dysfunction in RNA processing as a major pathogenic mechanism. However, whether a precise RNA pathway is particularly affected remains unknown. Evidence suggests that FUS, that is mutated in familial ALS, and SMN, the causative factor in Spinal Muscular Atrophy (SMA), cooperate to the same molecular pathway, i.e. regulation of alternative splicing, and that disturbances in SMN-regulated functions, either caused by depletion of SMN protein (as in the case of SMA) or by pathogenic interactions between FUS and SMN (as in the case of ALS) might be a common theme in both diseases. In this work, we followed these leads and tested their pathogenic relevance in vivo. FUS-associated ALS recapitulates, in transgenic mice, crucial molecular features that characterise mouse models of SMA, including defects in snRNPs distribution and in the alternative splicing of genes important for motor neurons. Notably, altering SMN levels by haploinsufficiency or overexpression does not impact the phenotypes of mouse or Drosophila models of FUS-mediated toxicity. Overall, these findings suggest that FUS and SMN functionally interact and that FUS may act downstream of SMN-regulated snRNP assembly in the regulation of alternative splicing and gene expression.

Cells robustly reprogram gene expression during stress generated by protein misfolding and aggregation. In this condition, cells assemble the bulk of mRNAs into translationally silent stress granules (SGs), while they sustain the translation of specific mRNAs coding for proteins that are needed to overcome cellular stress. Alterations of this process are deeply associated to neurodegeneration. This is the case of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by a selective loss of motor neurons. Indeed, impairment of protein homeostasis as well as alterations of RNA metabolism are now recognized as major players in the pathogenesis of ALS. In particular, evidence shows that defective mRNA transport and translation are implicated. Here, we provide a review of what is currently known about altered mRNA translation in ALS and how this impacts on the ability of affected cells to cope with proteotoxic stress.

Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown. Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine-synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.

A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS., (© 2015. Published by The Company of Biologists Ltd.)

Defective nectin-1 and -4 have been implicated in ectodermal dysplasia (ED) syndromes with variably associated features including orofacial and limb defects. In particular, nectin-1 mutations cause cleft lip/palate ED (CLPED1; OMIM#225060), whereas defective nectin-4 is associated with ED-syndactyly syndrome (EDSS1; OMIM#613573). Although the broad phenotypic overlap suggests a common mode of action of nectin-1 and -4, little is known about the pathogenic mechanisms involved. We report the identification of, to our knowledge, a previously undescribed nectin-4 homozygous p.Val242Met missense mutation in a patient with EDSS1. We used patient skin biopsy and primary keratinocytes, as well as nectin-4 ectopic expression in epithelial cell lines, to characterize functional consequences of p.Val242Met and p.Thr185Met mutations, the latter previously identified in compound heterozygosity with a truncating mutation. We show that nectin-4-altered expression perturbs nectin-1 clustering at keratinocyte contact sites and delays, but does not impede cell-cell aggregation and cadherin recruitment at adherens junctions (AJs). Moreover, trans-interaction of nectin-1 and -4 induces the activation of Rac1, a member of the Rho family of small GTPases, and regulates E-cadherin-mediated cell-cell adhesion. These data outline a synergistic action of nectin-1 and -4 in the early steps of AJ formation and implicate this interaction in modulating the Rac1 signaling pathway.

Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GlcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Histone acetylation plays essential roles in cell cycle progression, DNA repair, gene expression and silencing. Although the knowledge regarding the roles of acetylation of histone lysine residues is rapidly growing, very little is known about the biochemical pathways providing the nucleus with metabolites necessary for physiological chromatin acetylation. Here, we show that mutations in the scheggia (sea)-encoded Sea protein, the Drosophila ortholog of the human mitochondrial citrate carrier Solute carrier 25 A1 (SLC25A1), impair citrate transport from mitochondria to the cytosol. Interestingly, inhibition of sea expression results in extensive chromosome breakage in mitotic cells and induces an ATR-dependent cell cycle arrest associated with a dramatic reduction of global histone acetylation. Notably, loss of SLC25A1 in short interfering RNA (siRNA)-treated human primary fibroblasts also leads to chromosome breaks and histone acetylation defects, suggesting an evolutionary conserved role for Sea/SLC25A1 in the regulation of chromosome integrity. This study therefore provides an intriguing and unexpected link between intermediary metabolism and epigenetic control of genome stability.

CAP (c-Cbl associated protein)/ponsin belongs to a family of adaptor proteins implicated in cell adhesion and signaling. Here we show that CAP binds to and co-localizes with the essential endocytic factor dynamin. We demonstrate that CAP promotes the formation of dynamin-decorated tubule like structures, which are also coated with actin filaments. Accordingly, we found that the expression of CAP leads to the inhibition of dynamin-mediated endocytosis and increases EGFR stability. Thus, we suggest that CAP may coordinate the function of dynamin with the regulation of the actin cytoskeleton during endocytosis.

Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type I gamma, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.

ArgBP2, and its brain-specific splice variant, nArgBP2, are interactors and substrates of Abl/Arg tyrosine kinases and of the ubiquitin ligase Cbl. They are members of a family of adaptor proteins that colocalize with actin on stress fibers and at cell-adhesion sites, including neuronal synapses. We show here that their NH2-terminal region, which contains a sorbin homology domain domain, interacts with spectrin, and we identify binding proteins for their COOH-terminal SH3 domains. All these binding partners participate in the regulation of the actin cytoskeleton. These include dynamin, synaptojanin, and WAVE isoforms, as well as WAVE regulatory proteins. At least two of the ArgBP2/nArgBP2 binding partners, synaptojanin 2B and WAVE2, undergo ubiquitination and Abl-dependent tyrosine phosphorylation. ArgBP2/nArgBP2 knockdown in astrocytes produces a redistribution of focal adhesion proteins and an increase in peripheral actin ruffles, whereas nArgBP2 overexpression produces a collapse of the actin cytoskeleton. Thus, ArgBP2/nArgBP2 is a scaffold protein that control the balance between adhesion and motility by coordinating the function of multiple signaling pathways converging on the actin cytoskeleton.

Tuba is a 178kD protein containing four NH2-terminal SH3 domains, a central Dbl homology (DH) domain followed by a BAR domain, and two COOH-terminal SH3 domains. The four NH2-terminal SH3 domains bind the GTPase dynamin, a protein critical for the fission of endocytic vesicles. The DH domain functions as a CDC42-specific guanine nucleotide exchange factor and is unique among DH domains because it is followed by a BAR domain rather than a PH domain. The COOH-terminal SH3 domain binds directly to N-WASP and Ena/VASP proteins, key regulatory proteins of the actin cytoskeleton, and recruits a larger protein complex comprising additional actin regulatory factors. The properties of Tuba provide new evidence for a functional link between dynamin, endocytosis, and actin. The presence of a BAR domain, rather than a PH domain, may reflect its action at high curvature regions of the plasma membrane. Its multiple binding sites for dynamin generate an exceptionally high avidity for this GTPase and make the NH2-terminal region of Tuba a very useful tool for the one-step purification of dynamin.

Tuba is a novel scaffold protein that functions to bring together dynamin with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Tuba binds a variety of actin regulatory proteins, including N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1, and Ena/VASP proteins, via a C-terminal SH3 domain. Direct binding partners include N-WASP and Ena/VASP proteins. Forced targeting of the C-terminal SH3 domain to the mitochondrial surface can promote accumulation of F-actin around mitochondria. A Dbl homology domain present in the middle of Tuba upstream of a Bin/amphiphysin/Rvs (BAR) domain activates Cdc42, but not Rac and Rho, and may thus cooperate with the C terminus of the protein in regulating actin assembly. The BAR domain, a lipid-binding module, may functionally replace the pleckstrin homology domain that typically follows a Dbl homology domain. The properties of Tuba provide new evidence for a close functional link between dynamin, Rho GTPase signaling, and the actin cytoskeleton.

Membrane phosphoinositides control a variety of cellular processes through the recruitment and/or regulation of cytosolic proteins. One mechanism ensuring spatial specificity in phosphoinositide signalling is the targeting of enzymes that mediate their metabolism to specific subcellular sites. Phosphatidylinositol phosphate kinase type 1 gamma (PtdInsPKI gamma) is a phosphatidylinositol-4-phosphate 5-kinase that is expressed at high levels in brain, and is concentrated at synapses. Here we show that the predominant brain splice variant of PtdInsPKI gamma (PtdInsPKI gamma-90) binds, by means of a short carboxy-terminal peptide, to the FERM domain of talin, and is strongly activated by this interaction. Talin, a principal component of focal adhesion plaques, is also present at synapses. PtdInsPKI gamma-90 is expressed in non-neuronal cells, albeit at much lower levels than in neurons, and is concentrated at focal adhesion plaques, where phosphatidylinositol-4,5-bisphosphate has an important regulatory role. Overexpression of PtdInsPKI gamma-90, or expression of its C-terminal domain, disrupts focal adhesion plaques, probably by local disruption of normal phosphoinositide balance. These findings define an interaction that has a regulatory role in cell adhesion and suggest new similarities between molecular interactions underlying synaptic junctions and general mechanisms of cell adhesion.

We have developed a procedure to predict the peptide binding specificity of an SH3 domain from its sequence. The procedure utilizes information extracted from position-specific contacts derived from six SH3/peptide or SH3/protein complexes of known structure. The framework of SH3/peptide contacts defined on the structure of the complexes is used to build a residue-residue interaction database derived from ligands obtained by panning peptide libraries displayed on filamentous phage. The SH3-specific interaction database is a multidimensional array containing frequencies of position-specific contacts. As input, SH3-SPOT requires the sequence of an SH3 domain and of a query decapeptide ligand. The array, that we call the SH3-specific matrix, is then used to evaluate the probability that the peptide would bind the given SH3 domain. This procedure is fast enough to be applied to the entire protein sequence database. Panning experiments were performed to search putative specific ligands of different SH3 domains in a database of decapeptides, or in a database of protein sequences. The procedure ranked some of the natural partners of interaction of a number of SH3 domains among the best ligands of the approximately 5. 6x10(9) different decapeptides in the SWISSPROT database. We expect the predictive power of the method to increase with the enrichment of the SH3-specific matrix by interaction data derived from new complex structures or from the characterization of new ligands. The procedure was developed using the SH3 domain family as test case but its application can easily be extended to other families of protein domains (such as, SH2, MHC, EH, PDZ, etc.)., (Copyright 2000 Academic Press.)

Peptide libraries may be constructed by grafting, in vitro, random DNA sequences into a carrier gene and then introducing the degenerate hybrid coding sequence into an expression organism. This review will focus on phage display, which was the first expression organism for peptide library expression to be described and which still maintains predominance in this area because of its simplicity, minimal cost, ease of manipulation, power and robustness. Using phage as the host, a repertoire of random peptides can be expressed that may be searched by a variety of screening or selection procedures. By physically associating each element of the peptide library with its coding sequence, selection for a property of a specific peptide results in the enrichment of the corresponding gene thus facilitating its cloning and amplification. This review focuses on the construction and screening of peptide libraries displayed on filamentous phage capsid and only briefly discusses the display of proteins and protein domains.