Biology subjects

Ismail, S.

Publications and source records attributed to Ismail, S..

18 recordsLinked to original sources

bioRxiv2026

VASH1 phosphorylation regulates tubulin detyrosination via CNP/PRKG1α signaling in DRG neurons.

Functional neuronal circuit formation requires precise regulation of axon growth and branching through dynamic cytoskeletal remodeling. Although microtubule detyrosination/re-tyrosination critically controls microtubule dynamics, how extracellular signals regulate this process remains unclear. Here, we identify a direct link between C-type natriuretic peptide, protein kinase cGMP-dependent 1 (CNP/cGMP/PRKG1) signaling and microtubule detyrosination in dorsal root ganglion (DRG) neurons. CNP stimulation induces growth cone enlargement, increases tyrosinated tubulin within growth cones, and reduces detyrosinated tubulin along axons, effects abolished by PRKG1 inhibition. Pharmacological inhibition or genetic deletion of the detyrosinating enzyme VASH1 phenocopies CNP-induced growth cone remodeling. In vitro kinase assays identify VASH1 as a direct PRKG1 substrate and reveal a specific C-terminal serine phosphorylation site (serine 337) that suppresses VASH1-SVBP microtubule association and detyrosination activity. Together, these findings establish VASH1-SVBP as a key downstream effector linking extracellular guidance signaling to microtubule remodeling during DRG neuronal development.

cell biology↗

bioRxiv2026

Deep learning models detect neuromuscular disease and quantify functional impairment from video-derived biomechanics data

Measurements of human movement are critical for evaluating patients with neuromuscular diseases. However, current clinical tools are limited in sensitivity and can fail to detect the full spectrum of movement changes patients may exhibit. These limitations can obscure early signs of decline, delaying interventions that could preserve function and independence. OpenCap, a smartphone application that performs video-based markerless motion capture, can help address this gap by enabling scalable, out-of-lab measurements of human movement. Here, we present a method for both detecting disease and quantifying the severity of impaired movement from smartphone videos of patient motion. We used OpenCap to measure kinematics from 337 individuals with diagnoses spanning 12 rare, genetically-defined neuromuscular diseases and 78 age-matched healthy controls. Our dataset is the largest existing publicly available resource of body segment kinematics for this class of diseases. These data were collected in neuromuscular clinics during regular clinic visits as well as at conferences and community events across the United States. We trained deep learning models on these kinematic time series to differentiate between participants with neuromuscular disease and controls (AUROC = 0.97 [0.93-1.00]). We also developed a movement impairment severity score that correlates with both participant ratings of their own functional abilities ({rho} = -0.81, p < 0.001) and existing clinical tests of movement capabilities such as run speed ({rho} = -0.83, p < 0.001). Our methods for detecting disease and deriving quantitative biomarkers of movement impairment from smartphone videos have the potential to complement clinical assessments, inform screening protocols, and improve clinical trial endpoints.

bioengineering↗

bioRxiv2026

A bifunctional H/ACA snoRNP mediates both pseudouridylation and rRNA scaffolding during ribosome assembly

The early steps of eukaryotic large ribosomal subunit assembly remain poorly understood due to the structural flexibility of pre-60S intermediates, whose rRNA is extensively modified by small nucleolar RNPs (snoRNPs). Some snoRNPs, however, lack any modification function and instead scaffold ribosome assembly through largely unknown mechanisms. Here, we show that the H/ACA snoRNP snR37 integrates both modifying and scaffolding roles. Biochemical and structural analyses reveal a canonical H/ACA core that pseudouridylates a conserved uridine in the A site of the peptidyl transferase center, the catalytic heart of the 60S subunit. Additional RNA helices recruit non-core proteins, the Upa1-Upa2 heterodimer and Rbp95, which mediate stable snR37 association with pre-60S complexes. These proteins cooperate with the Npa1 rRNA chaperone complex to link four rRNA domains, thereby structurally organizing early pre-60S intermediates and promoting proper formation of the PTC. This dual organization establishes a paradigm for snoRNPs combining rRNA modification and scaffolding functions.

biochemistry↗

bioRxiv2025

NanoBlocks: creating fluorescent biosensors from affinity binders using competitive binding

Genetically-encoded fluorescent biosensors have revolutionized our understanding of complex systems by permitting the in situ observation of chemical activities. However, only a comparatively small set of chemical activities can be monitored, largely due to the need to identify protein domains that undergo conformational and/or association changes in response to a stimulus. Here, we present a strategy that can convert simple affinity binders such as nanobodies into biosensors for their innate targets by introducing a peptide sequence that competes for the binding site. We demonstrate proof-of-concept implementations of this NanoBlock design, developing sensors based on the ALFA nanobody and on the PDZ domain of Erbin. We show that these sensors can reliably detect their targets in vitro, in mammalian cells, and as part of fluorescence-activated cell sorting (FACS) experiments. In doing so, our strategy offers a way to strongly expand the range of cellular processes that can be probed using fluorescent biosensors.

biochemistry↗

bioRxiv2025

Selective reduction of KCNA4 in vulnerable glutamatergic-serotonin neurons of the dorsal raphe nucleus in Alzheimers Disease

INTRODUCTIONWe previously demonstrated that htau mice recapitulate many neuropsychiatric features of early Alzheimers disease (AD), and that the dorsal raphe nucleus (DRN) contains distinct subregions. Herein, we investigate vulnerability of the centromedial DRN to pathologically-phosphorylated tau (pTau), a region composed predominantly of dually serotonergic/glutamatergic (5HT/glut) neurons. METHODSWe use computational, molecular, biophysical, and behavioral techniques to assess the centromedial DRN across preclinical and post-mortem settings. RESULTSThe centromedial DRN contains 5HT/glut neurons that differentially express ion-channel genes in the htau mouse. 5HT/glut neurons exhibit increased excitability, which we demonstrate may dually promote pTau accumulation and the severity of depressive-like behaviors in htau mice. At Braak 2, KCNA4 is reduced in 5HT/glut neurons in AD, which are especially vulnerable to pTau compared to 5HT-nonglut neurons. DISCUSSIONTau-mediated dysfunction of the DRN may be driven by changes in ion channel activity that concomitantly promote the spread of pTau in Braak progression.

neuroscience↗

bioRxiv2025

Development of a genetically encoded and potent PDE6D inhibitor

PDE6D is a trafficking chaperone of prenylated proteins, such as small GTPases. Several small molecule inhibitors have been developed against it, given that the oncogene K-Ras is one of the cargo proteins. Inhibitor development suffered from the fact that inhibitors against the hydrophobic pocket of PDE6D were typically poorly water-soluble. Here we describe the development of genetically encoded inhibitors that are inspired by high-affinity natural cargo of PDE6D. Our most potent inhibitor, SNAP-STI, encodes merely a farnesylated tetra-peptide, which efficiently blocks PDE6D binding of farnesylated cargo. Direct comparison with small molecule PDE6D inhibitors suggests its higher potency. We show that inhibition of K-Ras membrane anchorage and K-RasG12C-dependent MAPK-signaling by SNAP-STI is weak, consistent with what is observed after PDE6D knockdown. Our data therefore further support that PDE6D is not a suitable surrogate target for efficient inhibition of K-Ras membrane anchorage and MAPK-activity. Nonetheless, by exploiting contacts at the pocket entry, we established a generalizable strategy to design high-affinity PDE6D inhibitors, providing powerful tools for PDE6D biology and target validation.

biochemistry↗

bioRxiv2025

Probing direct interactions between nuclear proteins in cells with nxReLo

Nearly all biological processes depend on protein-protein interactions (PPIs). While various methods exist to study these interactions, investigating those that involve nuclear proteins, including structurally complex proteins containing long disordered regions, remains a significant challenge. Here, we developed nxReLo, a simple and fast cell culture-based colocalization assay, designed to identify and characterize interactions between nuclear proteins. PIWI-interacting RNAs (piRNAs) safeguard germline genome integrity, and the PPIs that facilitate piRNA production are therefore essential for animal reproduction, yet remain incompletely understood. We used nxReLo to investigate interactions between members of the Drosophila melanogaster Rhino-Deadlock-Cutoff (RDC) complex and two associated components, Bootlegger and Moonshiner, a nuclear protein network required for piRNA expression. We demonstrate the utility of the nxReLo assay by systematically screening pairwise interactions within the RDC network and by assembling a multiprotein complex from multiple components. By combining nxReLo assays with AlphaFold structural prediction, we characterized the Cutoff-Deadlock and the Bootlegger-Deadlock complexes in detail, providing molecular and structural insights. Specifically, we refined the domains involved in the interaction and identified interface point mutations that interfered with complex formation, validating the predicted structures. In conclusion, nxReLo facilitates rapid and simple testing of direct interactions between nuclear proteins in a cellular context, which is particularly important when working with structurally challenging proteins or when established interaction assays prove unsuccessful.

biochemistry↗

bioRxiv2025

Expanding the Tubulin Code: TTLL11 Polyglutamylase Drives Elongation of Primary Tubulin Chains

Microtubules (MTs) undergo diverse post-translational modifications that regulate their structural and functional properties. Among these, polyglutamylation - a dominant and conserved modification targeting the unstructured tubulin C-terminal tails - plays a pivotal role in defining the tubulin code. Here, we uncovered a novel mechanism by which tubulin tyrosine ligase-like 11 (TTLL11) expands and diversifies the code. Cryo-electron microscopy revealed a unique bipartite MT recognition strategy wherein TTLL11s binding and catalytic domains engage adjacent MT protofilaments. Biochemical assays identified previously unknown polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of - and {beta}-tubulin, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data firmly established a crosstalk between TTLL11-mediated polyglutamylation and other tubulin-modifying processes, notably the detyrosination/tyrosination cycle. This discovery unveils an unrecognized layer of complexity within the tubulin code and offers new insights into the molecular basis of functional specialization of cytoskeleton across diverse cellular contexts.

biochemistry↗

bioRxiv2024

A new insight into the role of CART peptide in serotonergic function and anxiety

Cocaine and amphetamine-regulated transcript (CART) peptide has been established as a contributor to anxiogenic behavior. Genetic mutations in the CART gene are associated with anxiety and depression, and increased CART expression has been reported in suicide victims. Extensive research has focused on the role of CART peptide in mesolimbic neurocircuitry, but its involvement in the dorsal raphe nucleus (DRN) and serotonin (5HT) system remains unexplored. Here we demonstrate that CART processes are proximal to 5HTDRN neurons and that microinjection of CART (55-102) peptide into the DRN has an anxiogenic effect in mice. Furthermore, central CART administration reduced cfos activation in 5HT neurons of the ventral DRN, which is a putative reward/anti-stress circuit. The inhibitory effect of CART on 5HTDRN neuronal function and local 5HT release is further demonstrated with in vivo fiber photometry coupled with calcium and 5HT biosensors and by mass spectrometry. Moreover, using Cre-dependent retrograde tracing, we observed DRN-projecting CART neurons in the Edinger Westphal nucleus (EW), nucleus accumbens (NAc), and various hypothalamic nuclei including the ventromedial hypothalamus (VMH). Interestingly, based on ex vivo electrophysiological recordings, acute stress increased excitability in DRN-projecting CART neurons located in the EW, but not in the VMH or NAc. This suggests that the stress may promote anxiety-like behavior by activating the EWCART[->]5HTDRN circuit that ultimately inhibits 5HT transmission. In sum, understanding the intricate dynamics of the CARTergic and 5HTergic systems proves crucial in addressing 5HT-related dysfunctions, providing invaluable insights into both health and disease.

neuroscience↗

bioRxiv2023

Phosphatases of regenerating liver balance F-actin rearrangements during T cell activation through WD repeat containing protein 1

Phosphatases of regenerating liver (PRLs) have been proposed to regulate actin dynamics in lymphoid cells. However, the mechanism mediating this role remained unknown. Here we showed the interaction of the PRLs with the actin regulator WD repeat containing protein 1 (WDR1). The interaction of the PRLs with WDR1 was dependent on F-actin integrity and the proper recruitment of the PRLs to cell membranes through the CAAX motif. Endogenous PRLs and WDR1 were distributed to the Immunological Synapse (IS) and perturbed expression of PRL-1 or PRL-2 by CRISPR/Cas9 mediated genome editing showed that the PRLs were required for proper distribution of WDR1 to filamentous (F)-actin at the IS. Further, perturbed expression of the PRLs or WDR1 resulted in defective IS assembly with deregulated F-actin rearrangements, altered positioning of LFA-1, and reduced IL-2 production. Interestingly, balanced expression of PRLs was required for accumulation of CD3{varepsilon} at the IS and early activating signalling. We propose that PRLs regulate early T cell signalling and are required for proper F-actin dynamics by regulating the WDR1 access to F-actin networks. As a consequence, PRLs regulate LFA-1 positioning at the IS and proper cytokine secretion.

immunology↗

bioRxiv2023

Temperature and intraspecific variation affect host-parasite interactions

Parasites play key roles in regulating aquatic ecosystems, yet the impact of climate warming on their ecology and disease transmission remains poorly understood. Isolating the effect of warming is challenging as transmission involves multiple interacting species and potential intraspecific variation in temperature responses of one or more of these species. Here, we leverage a wide-ranging mosquito species and its facultative parasite as a model system to investigate the impact of temperature on host-parasite interactions and disease transmission. We conducted a common garden experiment measuring parasite growth and infection rates at seven temperatures using 12 field-collected parasite populations and a single mosquito population. We find that both free-living growth rates and infection rates varied with temperature, which were highest at 18-24.5{degrees}C and 13{degrees}C, respectively. Further, we find intraspecific variation in peak performance temperature reflecting patterns of local thermal adaptation--parasite populations from warmer source environments typically had higher thermal optima for free-living growth rates. For infection rates, we found a significant interaction between parasite population and nonlinear effects of temperature. These findings underscore the need to consider both host and parasite thermal responses, as well as intraspecific variation in thermal responses, when predicting the impacts of climate change on disease in aquatic ecosystems.

ecology↗

bioRxiv2021

Influence of Pink Pigmented Facultative methylotrophic bacteria (PPFM) as a foliar spraying on the growth and productivity of strawberry(Fragaria xananassa Duch.).

Pink pigmented facultative methylotrophic bacterium (PPFM) has a favorable impact on plant development and production, it is known as a biostimulator, biofertilizer and biocontroller. Here we investigate the effect of foliar spraying of PPFM, 10% methanol,30% methanol, and their combinations on the growth, fruit quality, and yield of two strawberry cultivars. PPFM was isolated from cotton leaves using imprinting technique. 16S rRNA sequence analysis identified it to be Methylobacterium radiotolerance. Its 16S rRNA sequence were deposited in the Gene Bank under accession number MT644122.1. Two field experiments were conducted during the 2017/2018 and 2018/2019 seasons to investigate the effect of foliar spraying of PPFM and methanol (10 and 30%) on the growth, fruit quality, and yield of two strawberry cultivars. The obtained results showed that, there were no significant differences in the most characteristics between the two cultivars except foliage fresh weight and early yield were higher in cv. Florid, however, Festival cv. recorded higher total yield /plant, anthocyanins and ascorbic acid content in both seasons. Spraying PPFM exhibited the highest values of chlorophyll, fresh weight, total yield and quality. Furthermore, PPFM combined with methanol 10% gave the highest values of leaf area, dry matter %, early yield and some fruit quality. Spraying cv. Florida with PPFM resulted in the best interactions for early yield. However, the best interaction for total yield and most fruit quality features was observed with Festival c.v. and spraying PPFM. It is reasonable to conclude that PPFM is the most effective treatment, increasing strawberry total yield/fed by 28.1 % in the 1st season and 27.91 % in the 2nd season compared to the control.

microbiology↗

bioRxiv2020

CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration

The Scar/WAVE complex is the major driver of actin nucleation at the plasma membrane, resulting in lamellipodia and membrane ruffles. While lamellipodia aid migration, membrane ruffles can generate macropinosomes - cup-like structures - important for nutrient uptake and regulation of cell surface receptor levels. How macropinosomes are formed and the role of the actin machinery in their formation and resolution is still not well understood. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A has not been characterised. Here we implicate CYRI-A as a key regulator of macropinosome maturation and integrin internalisation from the cell surface. We find that CYRI-A is recruited to nascent macropinosomes in a transient but distinct burst, downstream of PIP3-mediated RAC1 activation and the initial burst of actin assembly driving cup formation, but upstream of internalisation and RAB5 recruitment to the macropinosome. Together, our data place CYRI-A as a local suppressor of actin dynamics, enabling the resolution of the macropinocytic cup. The failure of CYRI-depleted cells to resolve their macropinocytic cups results in reduced integrin a5b1 internalisation, leading to enhanced spreading, invasive behaviour and anchorage-independent 3D growth. We thus describe a new role for CYRI-A as a highly dynamic regulator of RAC1 activity at macropinosomes, modulating homeostasis of integrin surface presentation, with important functional consequences.

cell biology↗

bioRxiv2020

Complete genome sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 unveils a large multidrug resistant plasmid encoding the NDM-1 and OXA-58 carbapenemases

Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125 whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3 and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids whereas pAC1633-2 is a 12,651 bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.

microbiology↗

bioRxiv2020

Immuno-informatics Characterization SARS-CoV-2 Spike Glycoprotein for Prioritization of Epitope based Multivalent Peptide Vaccine

The COVID-19 pandemic caused by SARS-CoV-2 is a public-health emergency of international concern and thus calling for the development of safe and effective therapeutics and prophylactics particularly a vaccine to protect against the infection. SARS-CoV-2 spike glycoprotein is an attractive candidate for vaccine, antibodies and inhibitor development because of many roles it plays in attachment, fusion and entry into the host cell. In this study, we characterized the SARS-CoV-2 spike glycoprotein by immune-informatics techniques to put forward potential B and T cell epitopes, followed by the use of epitopes in construction of a multi-epitope peptide vaccine construct (MEPVC). The MEPVC revealed robust host immune system simulation with high production of immunoglobulins, cytokines and interleukins. Stable conformation of the MEPVC with a representative innate immune TLR3 receptor was observed involving strong hydrophobic and hydrophilic chemical interactions, along with enhanced contribution from salt-bridges towards inter-molecular stability. Molecular dynamics simulation in solution aided further in interpreting strong affinity of the MEPVC for TLR3. This stability is the attribute of several vital residues from both TLR3 and MEPVC as shown by radial distribution function (RDF) and a novel analytical tool axial frequency distribution (AFD). Comprehensive binding free energies estimation was provided at the end that concluded major domination by electrostatic and minor from van der Waals. Summing all, the designed MEPVC has tremendous potential of providing protective immunity against COVID-19 and thus has the potential to be considered in experimental studies.

bioinformatics↗

bioRxiv2019

Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. We developed 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by spatially enriching cortical phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants restrict PI(3,4,5)P3 production to discrete cortical domains to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PIP3-signalling, and predicts long-term poor outcome across multiple cancers. Spatial enrichment of phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis. HighlightsO_LISpatial enrichment of PI(3,4,5)P3 is a molecular switch to promote invasion. C_LIO_LIIQSEC1 is a GEF for ARF5/6, promoting PIP5K-dependent PI(3,4,5)P3 production downstream of the HGF receptor Met. C_LIO_LIPro-invasive IQSEC1 variants restrict cortical PI(3,4,5)P3 production to subdomains that convert into invasive protrusions. C_LIO_LIIQSEC1 inhibition attenuates in vitro invasion and metastasis in vivo. C_LIO_LIIQSEC1 module is associated with poor outcome across tumour types. C_LI

cancer biology↗

bioRxiv2019

WASP restricts active Rac to maintain cells’ front-rear polarisation

Efficient motility requires polarised cells, with anterior pseudopods and a retracting rear. This polarisation requires that the pseudopod catalyst Rac is restricted to the front. Here we show that the Arp2/3 complex regulator WASP is important for maintaining front-rear polarity, using a mechanism that limits where active Rac localises. Dictyostelium cells lacking WASP inappropriately activate Rac and SCAR/WAVE at their rears, leading to reduced cell speed. WASP facilitates the internalisation of clathrin-coated pits, and its Rac-binding CRIB motif is considered essential for its localisation and activity. However, WASP mutants with deleted CRIB domains, or harbouring a new mutation that prevents Rac binding, localise normally, recruit Arp2/3 complex, and drive actin polymerisation. Similarly, Rac inhibitors do not block WASP localisation or activation. Despite this, WASP CRIB mutants cannot restore polarisation of active Rac. Thus, WASPs interaction with Rac regulates Rac activity and cell polarity, but is dispensable for activating actin polymerization.

cell biology↗

bioRxiv2017

CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a \"local inhibitor\" predicted and described in widely accepted mathematical models, but not previously identified in living cells.

cell biology↗