Comprehensive variant calling from whole-genome sequencing identifies a complex inversion that disrupts ZFPM2 in familial congenital diaphragmatic hernia.

BACKGROUND: Genetic disorders contribute to significant morbidity and mortality in critically ill newborns. Despite advances in genome sequencing technologies, a majority of neonatal cases remain unsolved. Complex structural variants (SVs) often elude conventional genome sequencing variant calling pipelines and will explain a portion of these unsolved cases. METHODS: As part of the Utah NeoSeq project, we used a research-based, rapid whole-genome sequencing (WGS) protocol to investigate the genomic etiology for a newborn with a left-sided congenital diaphragmatic hernia (CDH) and cardiac malformations, whose mother also had a history of CDH and atrial septal defect. RESULTS: Using both a novel, alignment-free and traditional alignment-based variant callers, we identified a maternally inherited complex SV on chromosome 8, consisting of an inversion flanked by deletions. This complex inversion, further confirmed using orthogonal molecular techniques, disrupts the ZFPM2 gene, which is associated with both CDH and various congenital heart defects. CONCLUSIONS: Our results demonstrate that complex structural events, which often are unidentifiable or not reported by clinically validated testing procedures, can be discovered and accurately characterized with conventional, short-read sequencing and underscore the utility of WGS as a first-line diagnostic tool.

The extent of Ssa1/Ssa2 Hsp70 chaperone involvement in nuclear protein quality control degradation varies with the substrate.

Protein misfolding is a recurring phenomenon that cells must manage; otherwise misfolded proteins can aggregate and become toxic should they persist. To counter this burden, cells have evolved protein quality control (PQC) mechanisms that manage misfolded proteins. Two classes of systems that function in PQC are chaperones that aid in protein folding and ubiquitin-protein ligases that ubiquitinate misfolded proteins for proteasomal degradation. How folding and degradative PQC systems interact and coordinate their respective functions is not yet fully understood. Previous studies of PQC degradation pathways in the endoplasmic reticulum and cytosol have led to the prevailing idea that these pathways require the activity of Hsp70 chaperones. Here, we find that involvement of the budding yeast Hsp70 chaperones Ssa1 and Ssa2 in nuclear PQC degradation varies with the substrate. In particular, nuclear PQC degradation mediated by the yeast ubiquitin-protein ligase San1 often involves Ssa1/Ssa2, but San1 substrate recognition and ubiquitination can proceed without these Hsp70 chaperone functions in vivo and in vitro. Our studies provide new insights into the variability of Hsp70 chaperone involvement with a nuclear PQC degradation pathway.

Vms1p is a release factor for the ribosome-associated quality control complex.

Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl-tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC.

Sterol Oxidation Mediates Stress-Responsive Vms1 Translocation to Mitochondria.

Vms1 translocates to damaged mitochondria in response to stress, whereupon its binding partner, Cdc48, contributes to mitochondrial protein homeostasis. Mitochondrial targeting of Vms1 is mediated by its conserved mitochondrial targeting domain (MTD), which, in unstressed conditions, is inhibited by intramolecular binding to the Vms1 leucine-rich sequence (LRS). Here, we report a 2.7 Å crystal structure of Vms1 that reveals that the LRS lies in a hydrophobic groove in the autoinhibited MTD. We also demonstrate that the oxidized sterol, ergosterol peroxide, is necessary and sufficient for Vms1 localization to mitochondria, through binding the MTD in an interaction that is competitive with binding to the LRS. These data support a model in which stressed mitochondria generate an oxidized sterol receptor that recruits Vms1 to support mitochondrial protein homeostasis.

The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control.

Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system. In the ubiquitin-proteasome system, ubiquitin-conjugating enzymes and ubiquitin ligases append polyubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount as a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, the reaction catalyzed by nuclear ubiquitin ligase San1 was reconstituted in vitro Our results demonstrate that San1 can function with two ubiquitin-conjugating enzymes, Cdc34 and Ubc1. Although Cdc34 and Ubc1 are both sufficient for promoting San1 activity, San1 functions preferentially with Ubc1, including when both Ubc1 and Cdc34 are present. Notably, a homogeneous peptide that mimics a misfolded PQC substrate was developed and enabled quantification of the kinetics of San1-catalyzed ubiquitylation reactions. We discuss how these results may have broad implications for the regulation of PQC-mediated protein degradation.

Means of self-preservation: how an intrinsically disordered ubiquitin-protein ligase averts self-destruction.

Ubiquitin-protein ligases (E3s) that ubiquitinate substrates for proteasomal degradation are often in the position of ubiquitinating themselves due to interactions with a charged ubiquitin-conjugating enzyme (E2). This can mediate the E3's proteasomal degradation. Many E3s have evolved means to avoid autoubiquitination, including protection by partner or substrate binding, preventative modifications, and deubiquitinating enzyme reversal of ubiquitination. Here we describe another adaptation for E3 self-protection discovered while exploring San1, which ubiquitinates misfolded nuclear proteins in yeast for proteasomal degradation. San1 is highly disordered in its substrate-binding regions N- and C-terminal to its RING domain. In cis autoubiquitination could occur if these flexible regions come in proximity to the E2. San1 prevents this by containing no lysines in its disordered regions; thus the canonical residue used for ubiquitin attachment has been selectively eliminated. San1's target substrates have lost their native structures and expose hydrophobicity. To avoid in trans autoubiquitination, San1 possesses little concentrated hydrophobicity in its disordered regions, and thus the that feature San1 recognizes in misfolded substrates has also been selectively eliminated. Overall the presence of key residues in San1 have been evolutionarily minimized to avoid self-destruction either in cis or in trans. Our work expands the ways in which E3s protect themselves from autoubiquitination.

Substrate recognition in nuclear protein quality control degradation is governed by exposed hydrophobicity that correlates with aggregation and insolubility.

Misfolded proteins present an escalating deleterious challenge to cells over the course of their lifetime. One mechanism the cell possesses to prevent misfolded protein accumulation is their destruction by protein quality control (PQC) degradation systems. In eukaryotes, PQC degradation typically proceeds via multiple ubiquitin-protein ligases that act throughout the cell to ubiquitinate misfolded proteins for proteasome degradation. What the exact feature of misfolding that each PQC ubiquitin-protein ligase recognizes in their substrates remains an open question. Our previous studies of the budding yeast nuclear ubiquitin-protein ligase San1 indicated that it recognizes exposed hydrophobicity within its substrates, with the threshold of hydrophobicity equivalent to that of 5 contiguous hydrophobic residues. Here, we uncover an additional parameter: the nature of the exposed hydrophobicity that confers San1-mediated degradation correlates with significant protein insolubility. San1 particularly targets exposed hydrophobicity that leads to insolubility and aggregation above a certain threshold. Our studies presented here provide additional insight into the details of misfolded nuclear protein recognition and demonstrate that there is selectivity for the type of exposed hydrophobicity.

A conserved deubiquitinating enzyme controls cell growth by regulating RNA polymerase I stability.

Eukaryotic ribosome biogenesis requires hundreds of trans-acting factors and dozens of RNAs. Although most factors required for ribosome biogenesis have been identified, little is known about their regulation. Here, we reveal that the yeast deubiquitinating enzyme Ubp10 is localized to the nucleolus and that ubp10Δ cells have reduced pre-rRNAs, mature rRNAs, and translating ribosomes. Through proteomic analyses, we found that Ubp10 interacts with proteins that function in rRNA production and ribosome biogenesis. In particular, we discovered that the largest subunit of RNA polymerase I (RNAPI) is stabilized via Ubp10-mediated deubiquitination and that this is required in order to achieve optimal levels of ribosomes and cell growth. USP36, the human ortholog of Ubp10, complements the ubp10Δ allele for RNAPI stability, pre-rRNA processing, and cell growth in yeast, suggesting that deubiquitination of RNAPI may be conserved in eukaryotes. Our work implicates Ubp10/USP36 as a key regulator of rRNA production through control of RNAPI stability.

Selective destruction of abnormal proteins by ubiquitin-mediated protein quality control degradation.

Misfolded proteins are continuously produced in the cell and present an escalating detriment to cellular physiology if not managed effectively. As such, all organisms have evolved mechanisms to address misfolded proteins. One primary way eukaryotic cells handle the complication of misfolded proteins is by destroying them through the ubiquitin-proteasome system. To do this, eukaryotes possess specialized ubiquitin-protein ligases that have the capacity to recognize misfolded proteins over normally folded proteins. The strategies used by these Protein Quality Control (PQC) ligases to target the wide variety of misfolded proteins in the cell will likely be different than those used by ubiquitin-protein ligases that function in regulated degradation to target normally folded proteins. In this review, we highlight what is known about how misfolded proteins are recognized by PQC ubiquitin-protein ligases.

Exposed hydrophobicity is a key determinant of nuclear quality control degradation.

Protein quality control (PQC) degradation protects the cell by preventing the toxic accumulation of misfolded proteins. In eukaryotes, PQC degradation is primarily achieved by ubiquitin ligases that attach ubiquitin to misfolded proteins for proteasome degradation. To function effectively, PQC ubiquitin ligases must distinguish misfolded proteins from their normal counterparts by recognizing an attribute of structural abnormality commonly shared among misfolded proteins. However, the nature of the structurally abnormal feature recognized by most PQC ubiquitin ligases is unknown. Here we demonstrate that the yeast nuclear PQC ubiquitin ligase San1 recognizes exposed hydrophobicity in its substrates. San1 recognition is triggered by exposure of as few as five contiguous hydrophobic residues, which defines the minimum window of hydrophobicity required for San1 targeting. We also find that the exposed hydrophobicity recognized by San1 can cause aggregation and cellular toxicity, underscoring the fundamental protective role for San1-mediated PQC degradation of misfolded nuclear proteins.

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates.

Protein quality control (PQC) degradation systems protect the cell from the toxic accumulation of misfolded proteins. Because any protein can become misfolded, these systems must be able to distinguish abnormal proteins from normal ones, yet be capable of recognizing the wide variety of distinctly shaped misfolded proteins they are likely to encounter. How individual PQC degradation systems accomplish this remains an open question. Here we show that the yeast nuclear PQC ubiquitin ligase San1 directly recognizes its misfolded substrates via intrinsically disordered N- and C-terminal domains. These disordered domains are punctuated with small segments of order and high sequence conservation that serve as substrate-recognition sites San1 uses to target its different substrates. We propose that these substrate-recognition sites, interspersed among flexible, disordered regions, provide San1 an inherent plasticity which allows it to bind its many, differently shaped misfolded substrates.

Functional corticomuscular connection during reaching is weakened following stroke.

OBJECTIVE: To investigate the functional connection between motor cortex and muscles, we measured electroencephalogram-electromyogram (EEG-EMG) coherence of stroke patients and controls. METHODS: Eight healthy controls and 21 patients with shoulder and elbow coordination deficits were enrolled. All subjects performed a reaching task involving shoulder flexion and elbow extension. EMG of the anterior deltoid (AD) and brachii muscles (BB, TB) and 64-channel scalp EEG were recorded during the task. Time-frequency coherence was calculated using the bivariate autoregressive model. RESULTS: Stroke patients had significantly lower corticomuscular coherence compared with healthy controls for the AD and BB muscles at both the beta (20-30 Hz) and lower gamma (30-40 Hz) bands during the movement. BH procedure (FDR) identified a reduced corticomuscular coherence for stroke patients in 11 of 15 scalp area-muscle combinations. There was no statistically significant difference between stroke patients and control subjects according to coherence in other frequency bands. CONCLUSION: Poorly recovered stroke survivors with persistent upper-limb motor deficits exhibited significantly lower gamma-band corticomuscular coherence in performing a reaching task. SIGNIFICANCE: The study suggests poor brain-muscle communication or poor integration of the EEG and EMG signals in higher frequency band during reaching task may reflect an underlying mechanism producing movement deficits post-stroke.

Physiological Cost Index as a proxy measure for the oxygen cost of gait in stroke patients.

BACKGROUND: Stroke survivors can exhibit abnormally elevated oxygen consumption during walking. Therapeutic interventions can improve gait deficits and oxygen consumption. A practical measure of oxygen cost is not available. This study tested the usefulness of an indirect index of oxygen cost, the Physiological Cost Index, and the ability of this index to discriminate between healthy adults and stroke survivors. METHODS: The authors studied 17 subjects with stroke and 10 healthy control participants. Participants walked 10 minutes at their chosen comfortable speed on a treadmill. Oxygen consumption and heart rate data were collected. Primary measures were oxygen cost and the Physiological Cost Index. Secondary measures were age and gait speed. RESULTS: The Physiological Cost Index and oxygen cost had a good to excellent correlation (r = .83, P < .001) for subjects with stroke. Both oxygen cost and the Physiological Cost Index were comparable in detecting a significantly abnormal elevation for stroke survivors versus healthy adults (P = .003 and .002, respectively). Age was not correlated with oxygen cost, the Physiological Cost Index, or chosen gait speed. A moderate correlation of gait speed to both the Physiological Cost Index and oxygen cost was found. CONCLUSIONS: The Physiological Cost Index can be used as a proxy index for the oxygen cost of walking in subjects after stroke because it is correlated with oxygen cost and is comparable to oxygen cost in its capability to discriminate between healthy controls and subjects with stroke. The Physiological Cost Index can be performed inexpensively on a routine basis in a clinical environment.

Intra-limb coordination deficit in stroke survivors and response to treatment.

PURPOSE: Purpose one was to characterize the consistency of intra-limb hip/knee (H/K) coordination according to a measure of average coefficient of correspondence (ACC) across strides. Purpose two was to investigate H/K ACC validity and ability to discriminate pre-/post-treatment change in stroke survivors. METHODS: Five healthy controls and 32 chronic (>12 mos) stroke survivors were enrolled, and H/K ACC was calculated for both groups. Comparison between controls and stroke was made using the Mann-Whitney Test. Convergent validity of H/K ACC was tested using the Pearson Correlation model with gait speed and the 6 min Walk Test (6MWT). Stroke survivors were randomized to either: (1) gait training with functional neuromuscular stimulation (FNS) using intramuscular (IM) electrodes or (2) gait training without FNS. Both groups had treatment 1.5 h/day, 5 days/week, for 12 weeks, including .5 h coordination exercise, .5 h body weight supported treadmill training (BWSTT), and .5 h over ground gait training. The FNS-IM group used FNS-IM for all treatment components; the No-FNS group did not. Pre-/post-treatment comparisons were made using ANOVA. RESULTS: H/K ACC detected a significant difference between controls versus stroke involved limb (p=.0001) and controls versus stroke uninvolved limb (p=.042). The H/K ACC measure was well-correlated with gait speed (r=.70) and 6MWT (r=.69). H/K ACC showed a significant treatment response to FNS-IM (p=.003), but not No-FNS (p=.747). CONCLUSIONS: H/K ACC sensitively discriminated between controls versus stroke involved or uninvolved limbs. H/K ACC was valid, with significant correlations with both walking speed and 6MWT. FNS-IM produced a significant gain in H/K ACC, and No-FNS did not.

A randomized controlled trial of functional neuromuscular stimulation in chronic stroke subjects.

BACKGROUND AND PURPOSE: Conventional therapies fail to restore normal gait to many patients after stroke. The study purpose was to test response to coordination exercise, overground gait training, and weight-supported treadmill training, both with and without functional neuromuscular stimulation (FNS) using intramuscular (IM) electrodes (FNS-IM). METHODS: In a randomized controlled trial, 32 subjects (>1 year after stroke) were assigned to 1 of 2 groups: FNS-IM or No-FNS. Inclusion criteria included ability to walk independently but inability to execute a normal swing or stance phase. All subjects were treated 4 times per week for 12 weeks. The primary outcome measure, obtained by a blinded evaluator, was gait component execution, according to the Tinetti gait scale. Secondary measures were coordination, balance, and 6-minute walking distance. RESULTS: Before treatment, there were no significant differences between the 2 groups for age, time since stroke, stroke severity, and each study measure. FNS-IM produced a statistically significant greater gain versus No-FNS for gait component execution (P=0.003; parameter estimate 2.9; 95% CI, 1.2 to 4.6) and knee flexion coordination (P=0.049). CONCLUSIONS: FNS-IM can have a significant advantage versus No-FNS in improving gait components and knee flexion coordination after stroke.

Response to upper-limb robotics and functional neuromuscular stimulation following stroke.

Twelve moderately to severely involved chronic stroke survivors (>12 mo) were randomized to one of two treatments: robotics and motor learning (ROB-ML) or functional neuromuscular stimulation and motor learning (FNS-ML). Treatment was 5 h/d, 5 d/wk for 12 wk. ROB-ML group had 1.5 h per session devoted to robotics shoulder and elbow (S/E) training. FNS-ML had 1.5 h per session devoted to functional neuromuscular stimulation (surface electrodes) for wrist and hand (W/H) flexors/extensors. The primary outcome measure was the functional measure Arm Motor Ability Test (AMAT). Secondary measures were AMAT-S/E and AMAT-W/H, Fugl-Meyer (FM) upper-limb coordination, and the motor control measures of target accuracy (TA) and smoothness of movement (SM). ROB-ML produced significant gains in AMAT, AMAT-S/E, FM upper-limb coordination, TA, and SM. FNS-ML produced significant gains in AMAT-W/H and FM upper-limb coordination.

Response of sagittal plane gait kinematics to weight-supported treadmill training and functional neuromuscular stimulation following stroke.

After stroke, persistent gait deficits cause debilitating falls and poor functional mobility. Gait restoration can preclude these outcomes. Sixteen subjects (>12 months poststroke) were randomized to two gait training groups. Group 1 received 12 weeks of treatment, 4 times a week, 90 min per session, including 30 min strengthening and coordination, 30 min over-ground gait training, and 30 min weight-supported treadmill training. Group 2 received the same treatment, but also used functional neuromuscular stimulation (FNS) with intramuscular (IM) electrodes (FNS-IM) for each aspect of treatment. Outcome measures were kinematics of gait swing phase. Both groups showed no significant pre-/posttreatment gains in peak swing hip flexion. Group 1 (no FNS) had no significant gains in other gait components at posttreatment or at follow-up. Group 2 (FNS-IM) had significant gains in peak swing knee flexion and mid-swing ankle dorsiflexion (p < 0.05) that were maintained for 6 months.