Biology subjects

Syed, A.

Publications and source records attributed to Syed, A..

17 recordsLinked to original sources

bioRxiv2025

Human CDK5-cyclin B1 structures uncover a conserved mitotic CDK activation mechanism

Cyclin-dependent kinase 5 (CDK5), long considered atypical and activated by non-cyclin cofactors in neurons, also functions in mitosis. To resolve its activation mechanism during mitosis, we determined the first high-resolution crystal structures of CDK5-cyclin B1 in apo and nucleotide-bound states. Contrary to AlphaFold predictions, we find CDK5 structures unexpectedly mirrors CDK1-cyclin B1 assembly and activation, establishing CDK5 as a bona fide mitotic kinase acting in parallel with CDK1.

biochemistry↗

bioRxiv2025

CHAMP1 Complex Promotes Heterochromatin Assembly and Reduces Replication Stress

Replication stress is a major driver of genomic instability and a hallmark of cancer cells. Although dynamic heterochromatin remodeling has been implicated in replication stress response, the precise mechanisms remain unclear. Here, we identify the CHAMP1 complex, composed of CHAMP1, POGZ, HP1, and the H3K9 methyltransferase SETDB1, as a critical regulator of heterochromatin assembly at stalled replication forks. Upon replication stress, the CHAMP1 complex is recruited to stalled forks where it facilitates H3K9me3 deposition, creating a repressive chromatin environment that shields replication forks from MRE11-mediated degradation. The complex promotes the recruitment of the origin recognition complex (ORC) to sites of replication stress, such as the telomeric heterochromatin in alternative lengthening of telomeres (ALT)-positive tumor cells, thereby supporting efficient telomeric DNA replication. Loss of CHAMP1 disrupts ORC2 recruitment and impairs fork restart, leading to increased micronuclei formation and heightened sensitivity to replication stress. Notably, CHAMP1 deficiency induces synthetic lethality with FANCM inhibition in ALT-positive tumor cells, and the CHAMP1 complex is essential for the survival of CCNE1-amplified ovarian cancers. These findings uncover a chromatin-based mechanism of replication fork stabilization and suggest that CHAMP1 may represent a candidate therapeutic vulnerability in cancers with elevated replication stress.

molecular biology↗

bioRxiv2024

CHAMP1 complex directs heterochromatin assembly and promotes homology-directed DNA repair

The CHAMP1 complex, a little-known but highly conserved protein complex consisting of CHAMP1, POGZ, and HP1, is enriched in heterochromatin though its cellular function in these regions of the genome remain unknown. Here we show that the CHAMP complex promotes heterochromatin assembly at multiple chromosomal sites, including centromeres and telomeres, and promotes homology-directed repair (HDR) of DNA double strand breaks (DSBs) in these regions. The CHAMP1 complex is also required for heterochromatin assembly and DSB repair in highly-specialized chromosomal regions, such as the highly-compacted telomeres of ALT (Alternative Lengthening of Telomeres) positive tumor cells. Moreover, the CHAMP1 complex binds and recruits the writer methyltransferase SETDB1 to heterochromatin regions of the genome and is required for efficient DSB repair at these sites. Importantly, peripheral blood lymphocytes from individuals with CHAMP1 syndrome, an inherited neurologic disorder resulting from heterozygous mutations in CHAMP1, also exhibit defective heterochromatin clustering and defective repair of local DSBs, suggesting that a defect in DNA repair underlies this syndrome. Taken together, the CHAMP1 complex has a novel role in heterochromatin assembly and the enhancement of HDR in heterochromatin.

molecular biology↗

bioRxiv2024

Isolation, identification, and characterisation of the malachite green detoxifying bacterial strain Bacillus pacificus ROC1 and the azoreductase AzrC

Malachite green (MG) is used as a dye for materials such as wood, cotton, and nylon, and is used in aquaculture to prevent fungal and protozoan diseases. However, it is highly toxic, with carcinogenic, mutagenic, and teratogenic properties, resulting in bans worldwide. Despite this, MG is still frequently used in many countries due to its efficacy and economy. MG is persistent in the environment and so requires degradative intervention. In this work we isolated Bacillus pacificus ROC1 strain from a salt flat in Pakistan that had the ability to aerobically detoxify MG, as determined by bacterio- and phyto-toxicity assays. We demonstrate immobilized B. pacificus ROC1 can effectively detoxify MG, which highlights a potential method for its biodegradation. Genomic sequencing identified three candidate azo-reductases within B. pacificus ROC1 that could be responsible for the MG-degrading activity. These were cloned, expressed and purified from Escherichia coli, with one (AzrC), catalyzing the reduction of MG to leuco-MG in vitro. AzrC was crystallised and MG was captured within the active site in a Michaelis complex, providing structural insight into the reduction mechanism. Altogether, this work identifies a bacterium capable of aerobically degrading a major industrial pollutant and characterizes the molecular basis for this activity.

biochemistry↗

bioRxiv2024

Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd

Haemophilus influenzae is a bacterial species of interest for its medical relevance and utility as a model system. Despite its role in several landmark molecular and synthetic biology studies, H. influenzae remains underexplored as a potential chassis organism. The limited availability of reliable and convenient transformation methods and genetic tools for H. influenzae are obstacles to this end. However, a strain of H. influenzae Rd KW20 lacking the type II restriction endonucleases HindII and HindIII has previously been developed. Here, we show that this strain is more readily transformable by electroporation than wild-type Rd KW20. We also developed a series of multi-host plasmids carrying antibiotic selection and fluorescent visual markers based on the pSU20 vector. The availability of H. influenzae {Delta}HindII/III, paired with the electroporation method and plasmids presented here, will promote the exploration of H. influenzae as a host organism for synthetic biology applications.

synthetic biology↗

bioRxiv2024

Development of subunit selective proteasome substrates for Schistosoma species

Schistosomiasis, or bilharzia, is a neglected tropical disease caused by Schistosoma spp. blood flukes that infects over 200 million people worldwide. Just one partially effective drug is available, and new drugs and drug targets would be welcome. The 20S proteasome is a validated drug target for many parasitic infections, including those caused by Plasmodium and Leishmania. We previously showed that anticancer proteasome inhibitors that act through the Schistosoma mansoni 20S proteasome (Sm20S) kill the parasite in vitro. To advance these initial findings, we employed Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to define the substrate cleavage specificities of the three catalytic {beta} subunits of purified Sm20S. The profiles in turn were used to design and synthesize subunit-specific optimized substrates that performed two to eight fold better than the equivalent substrates used to measure the activity of the constitutive human proteasome (c20S). These specific substrates also eliminated the need to purify Sm20S from parasite extracts - a single step enrichment was sufficient to accurately measure substrate hydrolysis and its inhibition with proteasome inhibitors. Finally, we show that the substrate and inhibition profiles for the 20S proteasome from the three medically important schistosome species are similar, suggesting that data arising from an inhibitor development campaign that focuses on Sm20S can be extrapolated to the other two targets with consequent time and cost savings.

biochemistry↗

bioRxiv2023

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

We developed a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-validation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

biochemistry↗

bioRxiv2023

Dynamics of the DYNLL1/MRE11 complex regulates DNA end resection and recruitment of the Shieldin complex to DSBs

Extent and efficacy of DNA end resection at DNA double strand break (DSB)s determines the choice of repair pathway. Here we describe how the 53BP1 associated protein DYNLL1 works in tandem with Shieldin and the CST complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1 where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1 predominantly in the G1 cells. Shieldin localization to DSBs is dependent on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be re-sensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

molecular biology↗

bioRxiv2022

An international scholastic network to generate LexA enhancer-trap lines for Drosophila

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and eleven insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

genetics↗

bioRxiv2020

Enhanced specificity of high sensitivity somatic variant profiling in cell-free DNA via paired normal sequencing: design, validation, and clinical experience of the MSK-ACCESS liquid biopsy assay

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 98% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

genomics↗

bioRxiv2020

Polymerase Theta Inhibition Kills Homologous Recombination Deficient Tumors

PARP inhibitors (PARPi) have become a new line of therapy for Homologous Recombination (HR)-deficient cancers. However, resistance to PARPi has emerged as a major clinical problem. DNA polymerase theta (POL{theta}) is synthetic lethal with HR and a druggable target in HR-deficient cancers. Here, we identified the antibiotic Novobiocin (NVB) as a specific POL{theta} inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo. NVB directly binds to the POL{theta} ATPase domain, inhibits its ATPase activity, and phenocopies POL{theta} depletion. BRCA-deficient tumor cells and those with acquired PARPi resistance are sensitive to NVB in vitro and in vivo. Increased POL{theta} expression levels predict NVB sensitivity. The mechanism of NVB-mediated cell death in PARPi resistant cells is the accumulation of toxic RAD51 foci, which also provides a pharmacodynamic biomarker for NVB response. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. One Sentence SummaryWe identified Novobiocin as a specific POL{theta} inhibitor that selectively kills naive and PARPi resistance HR-deficient tumors in vitro and in vivo.

cancer biology↗

bioRxiv2020

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

Although laccase has been recognized as a wonder molecule, and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its larger-scale applications. Hence the present research was aimed to select high yielding fungal strains and to optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. Aspergillus sp. HB_RZ4 produced a copious amount of laccase on under meso-acidophillic shaking conditions in a medium containing glucose and yeast extract. A 25 {micro}M of CuSO4 enhanced the enzyme yield. The enzyme was best purified on Sephadex G-100 column. Purified enzyme resembled with the laccase of A. flavus. Kinetics of purified enzyme revealed the high substrate specificity and good velocity of reaction with ABTS as substrate. The enzyme was stable over a wide range of pH and temperature. The peptide structure of the purified enzyme resembled with the laccase of A. kawachii IFO 4308. The fungus decolorized various dyes independent of the requirement of a laccase mediator system (LMS). Aspergillus sp. HB_RZ4 came out as a potent natural producer of laccase, it decolorized the dyes even in absence of LMS and thus can be used for bioremediation.

biochemistry↗

bioRxiv2019

Oncologic Therapy Shapes the Fitness Landscape of ClonalHematopoiesis

Clonal hematopoiesis (CH) is frequent in cancer patients and associated with increased risk of therapy related myeloid neoplasms (tMN). To define the relationship between CH, oncologic therapy, and tMN progression, we studied 24,439 cancer patients. We show that previously treated patients have increased rates of CH, with enrichment of mutations in DNA Damage Response (DDR) genes (TP53, PPM1D, CHEK2). Exposure to radiation, platinum and topoisomerase II inhibitors have the strongest association with CH with evidence of dose-dependence and gene-treatment interactions. We validate these associations in serial sampling from 525 patients and show that exposure to cytotoxic and radiation therapy imparts a selective advantage specifically in hematopoietic cells with DDR mutations. In patients who progressed to tMN, the clone at CH demarcated the dominant clone at tMN diagnosis. CH mutational features predict risk of therapy-related myeloid neoplasm in solid tumor patients with clinical implications for early detection and treatment decisions.

genetics↗

bioRxiv2019

Production, purification and evaluation of biodegrading potential of PHB depolymerase of Stenotrophomonas sp. RZS 7

There are numerous reports on PHB depolymerases produced by a wide variety of microorganisms isolated from various habitats, however, reports on PHB depolymerase isolated from plastic contaminated sites are scares. Thermophilic PHB polymerase produced by isolates obtained from plastic contaminated sites is expected to have better relevance for its application in plastic/bioplastic degradation. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS 7 isolated from a plastic contaminated site. The isolate produced extracellular poly-{beta}-hydroxybutyrate (PHB) depolymerase in the mineral salt medium at 30oC during 4 days of incubation under shake flask condition. Purification of the enzyme was carried out by three different methods using PHB as a substrate. Purification of PHB depolymerase by ammonium salt precipitation, column chromatography, and solvent purification method was successfully carried out. Among the purification method tested, the enzyme was best purified by column chromatography on Octyl-Sepharose CL-4B column with maximum (0.7993 U mg-1 ml-1) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae. Experiments on assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS 7. The results obtained in FTIR analysis, HPLC study and GC-MS analysis confirmed the biodegradation attempt in liquid medium by Stenotrophomonas sp. RZS 7. Changes in surface morphology of PHB film in soil burial as observed in FE SEM analysis confirmed the biodegradation of PHB. The isolate was capable of degrading PHB and resulted in 87.74% degradation. Higher rate of degradation under natural soil condition is the result of activity of soil microbes that complemented the degradation by Stenotrophomonas sp. RZS 7.

microbiology↗

bioRxiv2019

Thermophilic PHB depolymerase of Stenotrophomonas sp., an isolate from the plastic contaminated site is best purified on Octyl-Sepharose CL-4B

There are numerous reports on PHB depolymerases produced by a wide variety of microorganisms isolated from various habitats, however, reports on PHB depolymerase isolated from plastic contaminated sites are scares. Thermophilic PHB polymerase produced by isolates obtained from plastic contaminated sites is expected to have better relevance for its application in plastic/ bioplastic degradation. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS 7 isolated from a plastic contaminated site. The isolate produced extracellular poly-{beta}-hydroxybutyrate (PHB) depolymerase in the mineral salt medium at 30oC during 4 days of incubation under shake flask condition. Purification of the enzyme was carried out by three different methods using PHB as a substrate. Purification of PHB depolymerase by ammonium salt precipitation, column chromatography, and solvent purification method was successfully carried out. Among the purification method tested, the enzyme was best purified by column chromatography on Octyl-Sepharose CL-4B column with maximum (0.7993 U/mg/ml) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae.

microbiology↗

bioRxiv2019

AGO1x prevents dsRNA-induced interferon signaling to promote breast cancer cell proliferation

Initially reported for viral RNA, elongation of polypeptide chains beyond the stop codon (translational readthrough (TR)) also occurs on eukaryotic transcripts. TR diversifies the proteome and can modulate protein levels 1-6. Here we report that AGO1x, a conserved TR isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double stranded RNAs, the induction of the interferon response and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x localizes to the nucleus, in the vicinity of nucleoli. We identify a novel interaction of AGO1x with the Polyribonucleotide Nucleotidyltransferase 1, depletion of either protein leading to dsRNA accumulation and impaired cell proliferation. Our study thus uncovers a novel function of an Argonaute protein outside of the miRNA effector pathway, in buffering dsRNA-induced interferon responses. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study suggests a new direction for limiting tumor growth.

cell biology↗

bioRxiv2019

Production, purification, and kinetics of the poly-β-hydroxybutyrate depolymerase from Microbacterium paraoxydans RZS6: A novel biopolymer-degrading organism isolated from a dumping yard

Poly-{beta}-hydroxybutyrate (PHB) depolymerase can decompose biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, few reports have described PHB depolymerases based on isolates obtained from plastic-contaminated sites that reflect the potential of the source organism. In this study, we evaluated Microbacterium paraoxydans RZS6 as a producer of extracellular PHB depolymerase isolated from a plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using the polyphasic approach, i.e., 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters, and BIOLOG identification, and was found to hydrolyze PHB on minimal salt medium containing PHB as the only source of carbon. Both isolates produced PHB depolymerase at 30{degrees}C within 2 days and at 45{degrees}C within 4 days. The enzyme was purified most efficiently using an octyl-sepharose CL-4B column, with the highest purification yield of 6.675 U/mg/mL. The enzyme required Ce2+ and Mg2+ ions but was inhibited by Fe2+ ions and mercaptoethanol. Moreover, enzyme kinetic analysis revealed that the enzyme was a metalloenzyme requiring Mg2+ ions, with optimum enzyme activity at 45{degrees}C (thermophilic) and under neutrophilic conditions (optimum pH = 7). The presence of Fe2+ ions (1 mM) and mercaptoethanol (1000 ppm) completely inhibited the enzyme activity. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled that of PHB depolymerase from Aureobacterium saperdae. Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase isolated from a plastic-contaminated site in the municipal area of Shahada, Maharashtra, India.

microbiology↗