Biology subjects

Khawaled, S.

Publications and source records attributed to Khawaled, S..

5 recordsLinked to original sources

bioRxiv2025

The Wnt/APC destruction complex targets SREBP2 in a β-catenin-independent pathway to control cholesterol metabolism

APC, the core scaffold of the Wnt destruction complex, targets the transcriptional co-activator {beta}-catenin for proteolysis. There is no convincing evidence that APC directs degradation of other substrates. Using a reconstituted cytosolic extract-based system and complementary in vivo and cellular assays, we show that SREBP2, the master regulator of cholesterol biosynthesis, is a direct APC-AXIN1 substrate. APC-dependent SREBP2 degradation is conserved in Xenopus embryos, mouse colon, and human colorectal cancer cells and restricts SREBP2 target-gene expression, cholesterol synthesis, and tissue cholesterol levels. Mechanistically, APC and AXIN1 promote SREBP2 degradation via a conserved phosphodegron, which marks SREBP2 for ubiquitination by the E3 enzyme, FBXW7. Like {beta}-catenin, SREBP2 is stabilized by extracellular Wnt ligands; unlike {beta}-catenin, its regulation is independent of GSK3{beta} and CK1 and requires the entire APC mutational cluster region (MCR), whereas {beta}-catenin turnover can operate with only a partial MCR. These findings define a {beta}-catenin-independent branch of Wnt signaling that couples APC to sterol metabolism, providing a mechanistic rationale to target the mevalonate/SREBP2 axis in APC-mutant colorectal cancer.

biochemistry↗

bioRxiv2025

Programmable conjugative CRISPR interference targeting genotoxin in the gut

Among microbially derived metabolites that influence host disease, colibactin garners increasing attention for its roles in the rising incidence of early-onset colorectal cancer. Produced by pks Escherichia coli, colibactin is a potent genotoxin, yet no approved therapeutics directly suppress it. Here, we engineered a self-transmissible conjugative plasmid to deliver CRISPR interference (CRISPRi) into multiple pks strains. This system silences transcription of colibactin biosynthetic genes and abolishes pks E. coli genotoxicity without the resistance mutations associated with wild-type Cas9-mediated bacterial inhibition. In mice, conjugation-mediated CRISPRi reduces DNA damage and pks E. coli colonization while preserving commensal diversity. Importantly, the system also lowers tumorigenesis driven by pks E. coli and outperforms a pharmacologic inhibitor in a mouse colorectal cancer model. Finally, we extend this platform to silence a second pathogenic metabolite, establishing a translational strategy to neutralize diverse microbial metabolites and expanding the toolkit for programmable live biotherapeutics in the gut.

bioengineering↗

bioRxiv2024

Polyamines sustain epithelial regeneration in aged intestines by modulating protein homeostasis

Aging hampers the regenerative potential of intestinal epithelium across species including humans, yet the underlying causes remain elusive. Here, using proteomic and metabolomic profiling of intestinal tissues together with functional assays, we characterized the temporal dynamics of regeneration following injury induced by 5-fluorouracil, a commonly used chemotherapeutic agent. Comparison of regeneration dynamics in mice of different ages revealed the emergence of a proteostasis stress signature and increased levels of polyamines following injury exclusively in old epithelia. Mechanistically, we show that delayed regeneration is an intrinsic feature of aged epithelial cells that display reduced protein synthesis and accumulation of ubiquitylated proteins. Notably, dietary restriction followed by re-feeding prior to injury increases polyamine pathway activation, enhances protein synthesis, and restores the regenerative capacity of aged intestines. Our findings highlight promising epithelial targets for interventions aimed at tackling the decline in tissue repair mechanisms associated with aging.

molecular biology↗

bioRxiv2024

Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms

Background & AimsSingle-cell RNA sequencing (scRNA) has empowered many insights into gastrointestinal microenvironments. However, profiling human biopsies using droplet-based scRNA (D-scRNA) is challenging since it requires immediate processing to minimize epithelial cell damage. In contrast, picowell-based (P-scRNA) platforms permit short-term frozen storage before sequencing. We compared P- and D-scRNA platforms on cells derived from human colon biopsies. MethodsEndoscopic rectosigmoid mucosal biopsies were obtained from two adults and conducted D-scRNA (10X Chromium) and P-scRNA (Honeycomb HIVE) in parallel using an individuals pool of single cells (> 10,000 cells/participant). Three experiments were performed to evaluate 1) P-scRNA with cells under specific storage conditions (immediately processed [fresh], vs. frozen at -20C vs. -80C [2 weeks]); 2) fresh P-scRNA versus fresh D-scRNA; and 3) P-scRNA stored at -80C with fresh D-scRNA. ResultsSignificant recovery of loaded cells was achieved for fresh (80.9%) and -80C (48.5%) P-scRNA and D-scRNA (76.6%), but not -20C P-scRNA (3.7%). However, D-scRNA captures more typeable cells among recovered cells (71.5% vs. 15.8% Fresh and 18.4% -80C P-scRNA), and these cells exhibit higher gene coverage at the expense of higher mitochondrial read fractions across most cell types. Cells profiled using D-scRNA demonstrated more consistent gene expression profiles among the same cell type than those profiled using P-scRNA. Significant intra-cell-type differences were observed in profiled gene classes across platforms. ConclusionsOur results highlight non-overlapping advantages of P-scRNA and D-scRNA and underscore the need for innovation to enable high-fidelity capture of colonic epithelial cells. The platform-specific variation highlights the challenges of maintaining rigor and reproducibility across studies that use different platforms.

genomics↗

bioRxiv2018

MiR-16-1* and miR-16-2* possess strong tumor suppressive and anti-metastatic properties in osteosarcoma.

Osteosarcoma (OS) is an aggressive malignancy affecting mostly children and adolescents. MicroRNAs (miRNAs) play important roles in OS development and progression. Here we found that miR-16-1* and miR-16-2* \"passenger\" strands as well as the \"lead\" miR-16 strand possess strong tumor suppressive functions in human OS. We report different although strongly overlapping functions for miR-16-1* and miR-16-2* in OS cells. Ectopic expression of these miRNAs affected primary tumor growth, metastasis seeding, and chemoresistance and invasiveness of human OS cells. Loss-of-function experiments verified tumor suppressive functions of these miRNAs at endogenous levels of expression. Using RNA immunoprecipitation (RIP) assays, we identify direct targets of miR-16-1* and miR-16-2* in OS cells. Furthermore, validation experiments identified FGFR2 as a direct target for miR-16-1* and miR-16-2*. Overall, our findings underscore the importance of passenger strand miRNAs in osteosarcomagenesis.\n\nNovelty and ImpactOsteosarcoma (OS) can be a fatal disease. MicroRNAs (miRNAs) play crucial roles in osteosarcomagenesis. In this study, we identify miR-16-1* and miR-16-2* as strong tumor suppressors and anti-metastatic genes in OS. This is the first report demonstrating tumor suppressive functions of passenger strands of these miRNAs in OS. Given that MIR-16-1 is located in 13q14 region that is commonly deleted in several human malignancies, our findings shed light on oncogenic mechanisms triggered by 13q14 deletion.

cancer biology↗