Biology subjects

Kandel, R.

Publications and source records attributed to Kandel, R..

6 recordsLinked to original sources

bioRxiv2026

Structure of a key adhesin-exopolysaccharide interaction provides insights into matrix assembly in Vibrio cholerae biofilms

Biofilms serve as a protective mechanism for bacteria, including many pathogens. To form such communities, bacteria secrete macromolecules that form an extracellular matrix serving as a barrier against environmental threats, such as predation, antibiotics, and the host immune system. To be effective, a biofilm must anchor to foreign surfaces and retain sufficient stiffness, in the environment or in a host. However, how this matrix self-organizes to support biofilm formation remains a mystery at the molecular level. The human pathogen Vibrio cholerae produces biofilms primarily composed of an exopolysaccharide called VPS (Vibrio polysaccharide), consisting of an unusually-modified repeating tetrasaccharide core unit. VPS engages with two secreted adhesion proteins, Bap1 and RbmC, which adhere the biofilm to abiotic and biotic surfaces, and serve to strengthen the biofilm by interacting with VPS using a conserved {beta}-propeller. To pinpoint the interaction between the adhesins and purified segments of VPS, we determined the [~]1.6 [A] X-ray crystal structure of Bap1 bound to fragmented VPS and used the structure to carry out molecular dynamics simulations. The structure revealed a single binding site consisting of one tetrasaccharide unit involving an induced magnesium binding site. Unexpectedly, the tetrasaccharide adopted a bent state caused by a rotation of the glycosidic bond between the central two monosaccharide units. Using a combination of mutagenesis, light scattering, and in situ fluorescent microscopy, we demonstrate that Bap1 not only facilitates biofilm adhesion, but is also required for proper VPS organization, through the identified binding pocket. Our structure reveals for the first time the interaction between a biofilm exopolysaccharide and matrix protein, as well as insights into conformational changes of exopolysaccharide induced by this binding. Our findings provide a generalizable approach for studying the biophysical and biochemical properties of carbohydrate-dependent biofilm assembly, which may lead to new ways to treat disease caused by biofilm-forming bacterial pathogens by disrupting the exopolysaccharide-protein interactions.

microbiology↗

bioRxiv2026

Conformational dynamics of exopolysaccharides underlie biofilm matrix mechanics in Vibrio cholerae

Polysaccharides remain the least understood biomacromolecules, particularly in terms of the relationship between their chemical structure and physical properties. On the other hand, polysaccharides often serve as the main structural components in biofilms: surface-attached aggregates of bacterial cells encased within a mechanically resilient extracellular matrix. The large chemical space explored by bacteria within biofilms provides excellent opportunities to establish the structure-function relationship for polysaccharides. In this paper, we systematically characterize various polymer properties of Vibrio polysaccharide (VPS), the major exopolysaccharide in biofilms formed by Vibrio cholerae, the causative agent of pandemic cholera. Using a combination of shear rheology, dynamic and static light scattering, and small-angle X-ray scattering, we measure the viscosity, molecular weight, persistence length, radius of gyration, and hydrodynamic radius of this chemically unique biopolymer. Combining all-atom and coarse-grained simulations, we show how the conformational flexibility of a single glycosidic linkage within each VPS monomer can lead to dramatic compaction of the entire polymer chain and nonclassical entanglement behavior. Our comprehensive quantification represents a rare endeavor for bacterial biofilms, whose matrix composition and physical properties remain largely nebulous; it also represents a significant step towards a detailed understanding of the molecular origins of biofilm mechanics.

biophysics↗

bioRxiv2025

Generating 3D Models of Carbohydrates with GLYCAM-Web

The carbohydrate 3D structure-prediction tools (builders) at GLYCAM-Web (glycam.org) are widely used for generating experimentally-consistent 3D structures of oligosaccharides suitable for data interpretation, hypothesis generation, simple visualization, and subsequent molecular dynamics (MD) simulation. The graphical user interface (GUI) enables users to create carbohydrate sequences (e.g. DGalpb1-4DGlcpb1-OH) that are converted to 3D models of the carbohydrate structures in multiple formats, including PDB and OFF (AMBER software format). The resulting structures are energy minimized prior to download and online visualization. There are advanced options for selecting which shapes (rotamers) of the oligosaccharide to generate, and for creating explicitly solvated structures for subsequent MD simulation. The GLYCAM-Web builders integrate known conformational preferences of oligosaccharides, summarized here, and employ the GLYCAM forcefield for energy minimization with algorithms tailored for speed and scalability. Even for large oligosaccharides (100 residues, ~2100 atoms) a 3D structure is typically returned to the user in less than a minute.

biophysics↗

bioRxiv2022

Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNF and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNF exposure, at a concentration that can induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. Unlike iAF cells, NP cells treated with TNF accumulated more intracellular ROS and secreted more H2O2. Following TNF treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 M) induced senescence, however unlike TNF, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling, iAF and TNF-treated NP cells were co-cultured. In contact co-culture the NP cells did induce iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (TNF). Further studies will investigate if human disc cells respond similarly.

cell biology↗

bioRxiv2022

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER) where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated-degradation (ERAD) removes these clients from the ER membrane to the cytosol in a process known as retrotranslocation. Our recent work demonstrates that rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated integral membrane ERAD substrates. To survey for potential interaction partners of Dfm1, we performed protein-proximity labeling by BioID (proximity-dependent biotin identification) followed by mass spectrometry and identified several interacting proteins known to play a role in the sphingolipid biosynthesis pathway. Specifically, we found that Dfm1 physically interacts with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components and is critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. We demonstrate for the first time that Dfm1 has a role in ER export, a function that is independent of Dfm1s canonical ERAD retrotranslocation function. Specifically, we show that loss of Dfm1 results in the accumulation of phosphorylated Orm2 at the ER, suggesting a novel role for Dfm1 in controlling Orm2 export from the ER and its subsequent degradation by EGAD. Moreover, recruitment of Cdc48 by Dfm1, which is critical for its role in ERAD retrotranslocation, is dispensable for Dfm1s role in ER export. Given that the accumulation of human Orm2 homologs, ORMDLs, are associated with many maladies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

cell biology↗

bioRxiv2021

Phosphoproteomic analysis of chondrocytes after short-term exposure to inorganic polyphosphate

Osteoarthritis is a debilitating disease of the joint that affects over 230 million people worldwide. Currently there are no treatments that slow the progression of this disease. For these reasons, new biological treatment options are currently being explored. Inorganic polyphosphates are naturally occurring biological molecules that have an anabolic effect on chondrocytes grown in vitro in the presence of Ca2+. We hypothesized that when examining significant changes in protein phosphorylation, key candidates would emerge that could help to elucidate the anabolic effects of polyphosphate on chondrocytes. Therefore, we conducted a large-scale quantitative proteomic and phosphoproteomic study of bovine primary articular chondrocytes after 30-minute treatment with inorganic polyphosphate and Ca2+. Mass spectrometry identified more than 6000 phosphorylation sites on [~]1600 chondrocyte phosphoproteins while proteomic analysis detected approximately 4100 proteins. Analysis of the data revealed a swift and dynamic response to polyphosphate after 30 minutes. What emerged from the list of proteins most affected by the treatment were proteins with key roles in chondrogenesis including TNC, IGFBP-5, and CTGF, indicating that polyphosphate plays an important role in chondrocyte metabolism. This phosphoproteome serves as a meaningful resource to help elucidate the molecular events that contribute to extracellular matrix production in cartilage.

bioengineering↗