Biology subjects

Saad, H.

Publications and source records attributed to Saad, H..

8 recordsLinked to original sources

bioRxiv2026

Adaptive Charge Modulation Enables Focal, Selective Spinal Cord Stimulation

Clinical neuromodulation primarily employs near-field low frequency electrical stimulation to activate neurons in the immediate vicinity of the electrode. We introduce Adaptive Charge Modulation (ACM), a spatiotemporal, charge-balanced stimulation strategy that focuses activation at deep tissue sites distant from the stimulating contacts. We apply ACM to stimulate deep regions of the spinal cord, distant from dorsal root entry zones which are preferentially activated during low frequency stimulation (LFS). ACM applies multipolar, biphasic rectangular pulses to exceed activation thresholds in deeper neuronal populations, with reduced surface activation, potentially due to high-frequency suppression of neural activity. In epidural spinal cord stimulation in rats, ACM achieved single-muscle selectivity among fourteen monitored muscles with minimal co-activation of other muscles. Using simultaneous, high spatiotemporal resolution, 2,112-channel brain-spine recordings, we characterized the response latencies and pathways consistent with focal recruitment at depth. We observed chronic stability of the electrode and ACM in freely behaving animals over 68 days post-implantation. By enabling focal activation with epidural surface electrodes, ACM may expand the reach and precision of neuromodulation and neural interfaces.

bioengineering↗

bioRxiv2026

Mannosidases IA, IB and IC are in segregated vesicular structures and involved in both glycoprotein quality control and maturation

N-linked glycoprotein processing critically depends on the trimming of -1,2 mannose residues, a key step required both for glycoprotein maturation along the secretory pathway and for targeting defective glycoproteins to endoplasmic reticulum-associated degradation (ERAD). Mammalian cells express seven Class I -1,2 mannosidases, yet their individual roles remain poorly defined, particularly for ManIA, ManIB, and ManIC, which were originally considered Golgi-resident maturation enzymes. Here, we re-evaluated the subcellular localization and functional contributions of these three mannosidases to glycoprotein quality control and maturation. We found that ManIA, ManIB, and ManIC localize predominantly to quality control vesicles (QCVs), previously identified by our group, whereas only ManIC displays a substantial Golgi population. Surprisingly, each enzyme is confined to a different vesicular population. All three enzymes promote ERAD targeting of misfolded model glycoproteins, albeit with different substrate preferences. In addition, they redundantly support the maturation and cell-surface delivery of a model glycoprotein. Most strikingly, in vitro analyses revealed that ManIA, ManIB, and ManIC preferentially trim a properly folded model glycoprotein rather than its denatured form. This is the opposite of the substrate preference that we previously observed for ERManI, EDEM1, and EDEM2. These findings support a model in which ERManI and the EDEMs selectively process misfolded glycoproteins to promote their recognition by the proximal lectin OS-9 and subsequent ERAD. In contrast, ManIA, ManIB and ManIC can slowly process misfolded glycoproteins but act rapidly on properly folded glycoprotein molecules, releasing them from ER-Golgi lectin-mediated retention or retrieval pathways, thereby promoting forward trafficking and maturation in the Golgi.

cell biology↗

bioRxiv2026

ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD), a process requiring their exit from the ER and delivery to cytosolic proteasomes via the key retrotranslocation step. Conventional assays detect fully extracted substrates, overlooking early initiation events. Here, we use an Escherichia coli-derived Biotin Acceptor Peptide (BAP)-biotin ligase BirA system to detect ERAD substrates upon cytosolic exposure. Using the model ERAD substrate H2a, we show that cytosolic BirA selectively biotinylates luminal BAP tags during early cytosolic exposure, enabling detection of substrates engaging the mammalian retrotranslocation machinery independently of subsequent extraction. Combined with high-resolution expansion fluorescence microscopy, this approach reveals active initiation of ERAD at the specialized ER-derived quality control compartment (ERQC). Substrates become transiently exposed to the cytosol while remaining membrane-associated, followed by tight coupling of polyubiquitination, transmembrane segment extraction, deglycosylation and degradation. Functional perturbations indicate that mannose trimming enables initiation, whereas p97 and HRD1 support later extraction and ubiquitination.

cell biology↗

bioRxiv2025

Helical charge distribution at the transmembrane-luminal interface determines subcellular localization

Transmembrane protein localization is typically dictated by short cytosolic tail sequences, whereas luminal domain contribution remains less understood. Here, we identify a short luminal juxtamembrane peptide as a key determinant of subcellular localization for Class I -1,2 mannosidases--ERManI, ManIA, ManIB, and ManIC--members of the glycoside hydrolase 47 family involved in N-glycoprotein processing. We previously showed that ERManI and ManIA localize to specialized quality control vesicles (QCVs); we now find that ManIB also partially localizes to these vesicles, whereas ManIC is predominantly Golgi-localized. ERManI, ManIA, and ManIB share a conserved luminal juxtamembrane distribution of charged residues, which diverges in ManIC. Structural predictions suggest that this region maintains an -helical conformation, with the charge pattern oriented on one face. Site-directed mutagenesis that disrupted this charge pattern or altered its helical register (via alanine insertions) shifted localization between QCVs and the Golgi. Moreover, grafting this peptide onto an unrelated transmembrane protein, {beta}-1,3-galactosyltransferase, redirected it from the Golgi to QCVs. These findings suggest that a specific three-dimensional charge pattern at the transmembrane-luminal interface serves as a localization signal. Unlike canonical linear motifs, this mechanism relies on the structural arrangement, revealing a previously unrecognized mode of organelle targeting within the secretory pathway.

cell biology↗

bioRxiv2023

Bioinformatics-Guided Discovery of Biaryl-Tailored Lasso Peptides

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature an isopeptide bond and a distinct lariat fold. A growing number of secondary modifications have been described that further decorate lasso peptide scaffolds. Using genome mining, we have discovered a pair of lasso peptide biosynthetic gene clusters (BGCs) that include cytochrome P450 genes. Here, we report the structural characterization of two unique examples of (C-N) biaryl-containing lasso peptides. Nocapeptin A, from Nocardia terpenica, is tailored with Trp-Tyr crosslink while longipepetin A, from Longimycelium tulufanense, features Trp-Trp linkage. Besides the unusual bicyclic frame, longipepetin A receives an S-methylation by a new Met methyltransferase resulting in unprecedented sulfonium-bearing RiPP. Our bioinformatic survey revealed P450(s) and further maturating enzyme(s)-containing lasso BGCs awaiting future characterization.

biochemistry↗

bioRxiv2022

COP I and II dependent trafficking controls ER-associated degradation

Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in mammalian cells in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an established ERAD substrate, asialoglycoprotein receptor H2a, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increased retrotranslocation and ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.

cell biology↗

bioRxiv2020

Exon probe sets and bioinformatics pipelines for all levels of fish phylogenomics

Exon markers have a long history of use in phylogenetics of ray-finned fishes, the most diverse clade of vertebrates with more than 35,000 species. As the number of published genomes increases, it has become easier to test exons and other genetic markers for signals of ancient duplication events and filter out paralogs that can mislead phylogenetic analysis. We present seven new probe sets for current target-capture phylogenomic protocols that capture 1,104 exons explicitly filtered for paralogs using gene trees. These seven probe sets span the diversity of teleost fishes, including four sets that target five hyper-diverse percomorph clades which together comprise ca. 17,000 species (Carangaria, Ovalentaria, Eupercaria, and Syngnatharia + Pelagiaria combined). We additionally included probes to capture exon markers that have been commonly used in fish phylogenetics, despite some being flagged for paralogs, to facilitate integration of old and new molecular phylogenetic matrices. We tested these probes and merged new exon-capture sequence data into an existing data matrix of 1,105 exons and 300 ray-finned fish species. We provide an optimized bioinformatics pipeline to assemble exon capture data from raw reads to alignments for downstream analysis. We show that loci with known paralogs are at risk of assembling duplicated sequences with target-capture, but we also assembled many useful orthologous sequences. These probe sets are a valuable resource for advancing fish phylogenomics because they can be easily extracted from increasingly available whole genome and transcriptome datasets, and also may be integrated with existing PCR-based exon and mitochondrial datasets.

evolutionary biology↗

bioRxiv2017

Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

Spatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock.\n\nOne Sentence SummaryUnequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

systems biology↗