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1.
Protein Sci ; 29(6): 1535-1549, 2020 06.
Article in English | MEDLINE | ID: mdl-32285480

ABSTRACT

Genetic variation in the membrane trafficking adapter protein complex 4 (AP-4) can result in pathogenic neurological phenotypes including microencephaly, spastic paraplegias, epilepsy, and other developmental defects. We lack molecular mechanisms responsible for impaired AP-4 function arising from genetic variation, because AP-4 remains poorly understood structurally. Here, we analyze patterns of AP-4 genetic evolution and conservation to identify regions that are likely important for function and thus more susceptible to pathogenic variation. We map known variants onto an AP-4 homology model and predict the likelihood of pathogenic variation at a given location on the structure of AP-4. We find significant clustering of likely pathogenic variants located at the interface between the ß4 and N-µ4 subunits, as well as throughout the C-µ4 subunit. Our work offers an integrated perspective on how genetic and evolutionary forces affect AP-4 structure and function. As more individuals with uncharacterized AP-4 variants are identified, our work provides a foundation upon which their functional effects and disease relevance can be interpreted.


Subject(s)
Adaptor Protein Complex 4/chemistry , Adaptor Protein Complex 4/genetics , Adaptor Protein Complex 4/metabolism , Evolution, Molecular , Genetic Variation/genetics , Humans , Models, Molecular , Protein Conformation , Sequence Homology, Amino Acid
2.
PLoS Genet ; 14(4): e1007363, 2018 04.
Article in English | MEDLINE | ID: mdl-29698489

ABSTRACT

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-ß4-µ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as "AP-4 deficiency syndrome". In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the µ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder.


Subject(s)
Adaptor Protein Complex 4/deficiency , Adaptor Protein Complex 4/genetics , Autophagy-Related Proteins/metabolism , Membrane Proteins/metabolism , Spastic Paraplegia, Hereditary/genetics , Spastic Paraplegia, Hereditary/metabolism , Vesicular Transport Proteins/metabolism , Adaptor Protein Complex 4/chemistry , Adaptor Protein Complex Subunits/chemistry , Adaptor Protein Complex Subunits/deficiency , Adaptor Protein Complex Subunits/genetics , Animals , Axons/metabolism , Behavior, Animal/physiology , Brain/metabolism , Brain/pathology , Disease Models, Animal , Female , Humans , Huntingtin Protein/chemistry , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Neurons/metabolism , Protein Aggregates/genetics , Protein Aggregation, Pathological/genetics , Protein Aggregation, Pathological/metabolism , Receptors, Glutamate/metabolism , Spastic Paraplegia, Hereditary/pathology , Spinal Cord/metabolism , Spinal Cord/pathology , trans-Golgi Network/metabolism
3.
Traffic ; 17(4): 400-15, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26756312

ABSTRACT

The adaptor protein 4 (AP4) complex (ϵ/ß4/µ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 ß4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal ß4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the ß4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on ß4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.


Subject(s)
Adaptor Protein Complex 4/metabolism , Adaptor Protein Complex 4/chemistry , Adaptor Protein Complex 4/genetics , Binding Sites , HEK293 Cells , HeLa Cells , Humans , Point Mutation , Protein Binding
4.
J Biol Chem ; 290(52): 30736-49, 2015 Dec 25.
Article in English | MEDLINE | ID: mdl-26542808

ABSTRACT

The heterotetrameric (ϵ-ß4-µ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the ß4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the ß4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.


Subject(s)
Adaptor Protein Complex 4/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Adaptor Protein Complex 4/chemistry , Adaptor Protein Complex 4/genetics , Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/genetics , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Dimerization , Humans , Molecular Sequence Data , Sequence Alignment , trans-Golgi Network/metabolism
5.
PLoS One ; 9(2): e88147, 2014.
Article in English | MEDLINE | ID: mdl-24498434

ABSTRACT

Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (µ1-µ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in µ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the µ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of µ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type µ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered µ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of µ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non-canonical site of µ4.


Subject(s)
Adaptor Protein Complex 4/chemistry , Adaptor Protein Complex 4/metabolism , Adaptor Protein Complex mu Subunits/chemistry , Adaptor Protein Complex mu Subunits/metabolism , Amyloid beta-Protein Precursor/metabolism , Adaptor Protein Complex 4/genetics , Amino Acid Sequence , Amyloid beta-Protein Precursor/chemistry , Binding Sites , Breast Neoplasms/metabolism , Calorimetry , Crystallography, X-Ray , Female , Fluorometry , Glioma/metabolism , Humans , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation/genetics , Polymerase Chain Reaction , Protein Conformation , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Two-Hybrid System Techniques
6.
Dev Cell ; 18(3): 425-36, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20230749

ABSTRACT

Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. Herein, we report the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) with the mu4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances gamma-secretase-catalyzed cleavage of APP to the pathogenic amyloid-beta peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein.


Subject(s)
Adaptor Protein Complex 4/metabolism , Amyloid beta-Protein Precursor/metabolism , Adaptor Protein Complex 4/chemistry , Adaptor Protein Complex 4/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amino Acid Sequence , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/genetics , Binding Sites/genetics , Crystallography, X-Ray , Endosomes/metabolism , HeLa Cells , Humans , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Protein Subunits , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction
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