Researchers discovered why a specific genetic mutation causes intellectual disability and autism spectrum disorder in children


Northwestern Medicine scientists have discovered why a specific genetic mutation causes intellectual disability and autism spectrum disorder in children.

“We have solved an important piece of the puzzle in understanding how this mutation causes intellectual disabilities and mental illness,” said lead author Peter Penzes, director of the new Center for Autism and Neurodevelopment at Northwestern University Feinberg School of Medicine.

The paper will be published Dec. 5 in Neuron.

Northwestern scientists discovered genetic mutations in human patients in a gene called Usp9x result in the brain growing fewer synapses.

That’s because Usp9x protects another protein called ankyrin-G, whose role is to grow and stabilize synapses.

The developing brain needs to build lots of synapses between neurons so cells can communicate while the brain grows, and to learn.

But when Usp9x is mutated, it can’t stabilize the synapse-enhancing ankyrin-G. Thus, the would-be enhancer protein degrades and destabilizes, resulting in fewer synapses in the brain, scientists found.

Individuals with this mutation have developmental delay, difficulty learning, increased anxiety and hyperactivity.

In addition to ankyrin-G, Usp9x also protects several other important synapse-enhancing proteins, which when mutated also cause intellectual disability and autism.

Usp9x is a master-stabilizer of many key proteins essential for brain development and learning.

It is notable that severe mutations in ankyrin-G are also known to cause intellectual disability and autism.

Or, if a person inherits a less severe form of the mutation in ankyrin-G, their synapses develop relatively normally in childhood.

But during adolescence – when there is a big turnover of synapses as the brain matures – more of these vital neuron connectors are lost than normal. The result can be schizophrenia and bipolar disease.

A possible cancer drug connection

Interestingly, Usp9x and related proteins are also involved in cancer and have been of interest to the pharmaceutical industry.

Hence, some candidates from the cancer drug development process could potentially be used to target Usp9x to treat some forms of intellectual disability, autism, schizophrenia and bipolar disorder.

About the Center for Autism and Neurodevelopment at Northwestern

The Center’s mission is to spur interdisciplinary research collaborations aimed at understanding the biological bases of autism and related neurodevelopmental disorders and to facilitate the translation of this knowledge into new treatments.

Northwestern scientists discovered genetic mutations in human patients in a gene called Usp9x result in the brain growing fewer synapses.

Autism is a highly prevalent neurodevelopmental disorder.

According to the Centers for Disease Control and Prevention, one in 68 children are identified as having Autism Spectrum Disorder.

“Over the past few years, many genetic causes of autism and related disorders have been found, which could provide insight into its neurobiological bases,” Penzes said.

“The next major challenge is to understand the function of these genes in shaping the development of brain circuits and how their improper function may derail neurodevelopment.

These genes and neurodevelopmental processes could serve as targets for new drugs aimed at treating autism and related disorders.”

Penzes also is the Ruth and Evelyn Dunbar Professor of Psychiatry and Behavioral Sciences at Feinberg.

Other Northwestern authors are Sehyoun Yoon, Euan Parnell, Marc P. Forrest, Mike Piper, Lachlan Jolly, and Stephen A. Wood.

Funding: The research was supported by grant R01MH107182 from the National Institute of Mental Health of the National Institutes of Health.

USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability.

Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual β-hairpin insertion.

The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains.

Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains.

The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the β-hairpin insertion both play important roles in polyUb chain processing and linkage specificity.

Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition.

The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.

Deubiquitinases (DUBs) remove a key posttranslational modification by ubiquitin (Ub) from target proteins. Of the seven classes of DUBs, Ub-specific proteases (USPs) comprise the largest subfamily and account for over half of the DUBs encoded by the human genome (1).

The USPs are cysteine proteases characterized by a conserved USP catalytic domain (CD) fold, which resembles an overall hand-like structure with fingers, thumb, and palm subdomains. The core fold can be divided into six conserved boxes, where the N-terminal box 1 contains a Cys of the catalytic triad, and the C-terminal boxes 5 and 6 contain a His and an Asp/Asn residue, respectively (2). The USP CD architecture is structurally conserved, although insertions at the loop regions between the boxes can drastically alter the size of the USP CDs (300–800 residues) (13).

Previous structural and biochemical studies of USP CDs, both in apo forms and in complex with Ub substrates, have provided insights into the molecular mechanism governing the diversity of USP activity regulation and substrate specificity (34).

The most-studied USP7 is regulated by both its Ub substrate and its C-terminal Ub-like domains (56). Apo-USP7 adopts an inactive conformation, with loops near the active site blocking Ub binding to a misaligned catalytic triad.

The binding of Ub induces conformational changes that promote substrate engagement and realignment of the catalytic residues into a productive state (5). A C-terminal peptide of USP7 further activates the CD activity by binding to an adjacent activation cleft to stabilize the active conformation of USP7 (7).

Unlike other subfamilies of DUBs, USPs generally show a limited preference for a polyUb chain of different linkages. Only a few USP structures in complex with a diUb were reported (811). The crystal structures of the zebrafish CYLD CD in complex with M1- and K63-linked diUbs revealed the important contributions of the insertions to influence the proximal Ub binding (8).

A zinc finger (ZnF) motif in the fingers subdomain of USP21 CD accommodates the distal Ub to allow specificity for a linear polyUb chain, as well as a diUb-like modifier ISG15 (9). A recent structure of USP18 CD in complex with ISG15 provides a molecular basis for its unique specificity toward ISG15 instead of Ub (10).

More recently, the cocrystal structure of USP30 CD with K6-linked diUb revealed the engagement of a proximal Ub binding site responsible for K6-linkage specificity (11).

USP9X was first identified as the fat facets (faf) gene in Drosophila with key roles in eye and embryo development (12). Subsequent homolog analyses revealed a highly conserved protein from Drosophila to mammals (>90%) with important functions in both development and diseases (1314).

As the X-linked ortholog of faf, human USP9X escapes from X-chromosome inactivation. A highly related paralog, USP9Y (91% sequence identity), is found on the Y chromosome, the function of which remains uncharacterized. USP9X protein regulates numerous cellular processes, including cell polarity, apoptosis, migration, and stem cell self-renewal (15). The importance of USP9X is further highlighted by a growing list of identified substrates and binding partners, including E3 ligases ITCH, SMURF1, and MARCH7 (1518).

USP9X is up-regulated in cervical, colorectal cancers, as well as myeloma, and proposed as a therapeutic target for cancer treatment (1920). Dysregulation of USP9X is also linked to neurodevelopmental disorders and neurodegenerative diseases, including epilepsy, autism, Parkinson’s, and Alzheimer’s disease (2022). Multiple genetic studies have established the causal link between USP9X mutations and X-linked intellectual disability (152324). Despite the importance of USP9X, our understanding of USP9X structure and activity remains limited.

We determined the X-ray crystal structure of the USP9X CD and characterized its activity using a set of novel activity-based di- and triUb probes (ABPs) and revealed the role of key structural elements responsible for polyUb chain recognition and cleavage. Comparison of the structure of USP9X with those of other USPs in complex with diUbs suggests that USP9X CD harbors three potential sites (S2, S1, and S1′) that could bind to distal, central, and proximal Ubs of a polyUb chain, respectively.

A unique β-hairpin insertion in sequence box 6 contributes to the recognition of the proximal Ub in a linkage-specific manner. Furthermore, labeling with triUb ABPs revealed linkage-dependent endo- or exo-cleavage by USP9X. Unexpectedly, besides the catalytic cysteine, we also identified that a noncatalytic cysteine close to the catalytic cleft reacts with a K11-linked diUb probe, which implicates that USP9X samples the conformational space of diUb before a productive substrate engagement. Taking these data together, we propose a model in which the USP9X CD serves as an extensive platform to accommodate multiple Ub moieties of a polyUb chain to achieve linkage-specific cleavage.

Northwestern University
Media Contacts:
Marla Paul – Northwestern University
Image Source:
The image is in the public domain.

Original Research: Closed access
“Usp9X Controls Ankyrin-Repeat Domain Protein Homeostasis during Dendritic Spine Development”. Peter Penzes et al.
Neuron doi:10.1016/j.neuron.2019.11.003.


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