Biology subjects

Kapuganti, S. K.

Publications and source records attributed to Kapuganti, S. K..

7 recordsLinked to original sources

bioRxiv2026

DDX3X syndrome mutations lock DDX3X-RNA conformational states to drive persistent pathological condensation and neuronal death

DDX3X is a highly conserved RNA helicase associated with RNA metabolism, translation initiation, and deciding cell fate choices. Spontaneous mutations in DDX3X cause a rare genetic human disorder called DDX3X syndrome, showing a spectrum of neurodevelopmental and intellectual abnormalities. How missense mutations in DDX3X lead to aberrant cellular functions and pathological consequences is unclear. Here, we demonstrate that specific DDX3X syndrome missense mutations induce the formation of persistent, solid-like DDX3X stress granules by structurally altering the conformation of the DDX3X-RNA complex. Structural interrogation of DDX3X syndrome missense mutations revealed critical mutations that perturb the DDX3X-RNA complex, exhibit high clinical pathogenicity scores, and are associated with cancers. N and C-terminal mutations adjacent to the helicase domain of the DDX3X (F182V, I190S, T198P, L556S, and L559H) showed augmented stress granule (SG) assembly, and formation of persistent DDX3X-SGs in neuronal and non-neuronal cells. Intriguingly, these mutations altered the liquid-like properties of DDX3X-SGs, forming solid-like SGs. Mechanistically, these mutations drive the persistent DDX3X-SGs by either locking DDX3X in an open, RNA-bound conformation or by increasing the rigidity of the DDX3X-RNA complex (I190S; T198P), thereby conferring a loss of liquid-like properties. The persistent solid-like DDX3X-SGs preferentially promoted neuronal lytic cell death and did not affect translation. Also, the C-terminal L556S and L559H mutations, which form persistent granules, promoted the DDX3X-driven {beta}-amyloid aggregation. Our observations indicate that DDX3X syndrome mutations near the N- and C-termini promote the formation of solid-like DDX3X condensates, neuropathological aggregation, and neuronal cell death, which might underlie the disease pathogenesis in humans. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/702492v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@164b436org.highwire.dtl.DTLVardef@73741aorg.highwire.dtl.DTLVardef@1cb6fcforg.highwire.dtl.DTLVardef@119b652_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Structural dynamics of Zika Virus NS1 via a reductionist approach reveal the disordered nature of its beta roll domain in isolation

Flavivirus Non-structural 1 (NS1) protein performs multiple functions such as host immune evasion, interaction with complement system factors, membrane rearrangement, etc. Therefore, it is highly plausible that significant structural and folding dynamics of NS1 might play a role in its multifunctionality. The dimeric structures of NS1 of multiple flaviviruses, including Zika virus (ZIKV), are available. However, its domain-wise dynamics perspective has not been explored so far. Therefore, it is of utmost importance to understand the structural conformations of NS1 and its domains in isolation, possibly highlighting the implications on the overall NS1 protein dynamics. Here, we have employed extensively long molecular dynamic (MD) simulations to understand the role of monomer, dimer, and a reductionist approach in understanding the dynamics of the three structural domains (i.e., {beta}- roll, wing, and {beta}-ladder) in isolation. Further, we experimentally validated our findings using CD spectroscopy and confirmed the intrinsically disordered behavior of NS1 {beta}-roll in isolation and lipid mimetic environments. We also found that the {beta}-ladder domain is highly flexible during long simulations. Therefore, we believe this study may have implications for significant dynamics played by NS1 protein, specifically during oligomerization of NS1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/488568v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1b2928org.highwire.dtl.DTLVardef@12b0b68org.highwire.dtl.DTLVardef@f9da77org.highwire.dtl.DTLVardef@175fbc1_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic representation of the ZIKV NS1 protein and the models that we have used in this study.

biophysics↗

bioRxiv2022

The aggregation potential of Zika virus proteome

The ability of human encoded soluble proteins to convert into amyloid fibrils is now recognized as a generic phenomenon in several human illnesses. Typically, such disease causal proteins/peptides consist of aggregation-prone regions (APR) that make them susceptible to misfolding and assemble into highly ordered {beta}-sheet rich fibrils, distinct from their native soluble state. Here, we show that the zika virus (ZIKV) consists of several such aggregation prone hotspots spread across its entire proteome. Using a combination of high-accuracy prediction tools, we identified APRs in both structural and non-structural proteins of ZIKV. Furthermore, we have experimentally validated the bioinformatic results by subjecting the ZIKV proteins and peptides to artificial aggregation inducing environment. Using a combination of dye-based assays (ThT and ANS) and microscopy techniques (HR-TEM and AFM), we further characterized the morphological features of amyloid-like fibrils. We found that Envelope domain III (EDIII) protein, NS1 {beta}-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein to be highly aggregating in the experimental setup. Our findings also pave the way for an extensive and detailed functional analysis of these predicted APRs in the future to enhance our understanding of the role played by amyloids in the pathogenesis of flavivirus. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/485915v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1bedf4dorg.highwire.dtl.DTLVardef@364676org.highwire.dtl.DTLVardef@14bf54aorg.highwire.dtl.DTLVardef@1d4fc19_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes

The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV-2 proteins, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our results show that these peptides and proteins form aggregates via a nucleation-dependent mechanism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome. SignificanceThe aggregation of proteins is linked with human disease in a variety of ways. In the case of viral infections, one could expect that the aberrant aggregation of viral proteins may damage the host cells, and also that viral particles may trigger the misfolding and aggregation of host proteins, resulting in damage to the host organism. Here we investigate the aggregation propensity of SARS-CoV-2 proteins and show that many of them can form aggregates that are potentially cytotoxic. In perspective, these results suggest that a better understanding of the effects of viruses on the human protein homeostasis system could help future therapeutic efforts.

biophysics↗

bioRxiv2021

P53 TAD2 Domain (38-61) Forms Amyloid-like Aggregates in Isolation

In many cases, when cellular machinery is unable to restore changed protein conformations, they start sticking through exposed hydrophobic patches and form aggregates. A strong association between protein aggregation and Human diseases (such as Alzheimers, Parkinsons, and Huntingtons disease) is well proven. p53 is a transcription factor that is also known as the guardian of the genome associated with cellular processes such as DNA repair, apoptosis, senescence, control of cell cycle, stress signaling and cellular homeostasis. The loss of function mutations in p53 have been implicated in several cancers. Experimental evidences have proposed a possible link between cancer and protein aggregation in evidence of the implication of amyloidogenic mutant proteins in ten different types of cancer. Aggregation studies focusing on different P53 domains, mostly, the central core domain and its mutants under the influence of various environmental conditions and P53 TAD domain (1-63) have been reported. P53 TADs interact with diverse cellular factors to modulate the function of P53 and elicit appropriate cellular response under different stress conditions. In this study, the aggregation of P53 TAD2 domain (38-61) have been studied in isolation. The aggregates were generated in-vitro in acidic pH conditions after in-silico scoring for amyloidogenic propensity and characterized using dye-based assays (ThT and bis-ANS fluorescence), CD spectroscopy, and microscopy (SEM, TEM and AFM). It was observed that P53 TAD2 follows nucleation-dependent kinetics and forms amyloid-like aggregates. On reductionists approach, this study highlights the nature of P53 TAD2 domain (amino acids 38-61) aggregation.

biochemistry↗

bioRxiv2020

Environmental Dependence of the Structure of the C-terminal Domain of the SARS-CoV-2 Envelope Protein

The SARS-CoV-2 envelope protein (E) is involved in a broad spectrum of functions in the cycle of the virus, including assembly, budding, envelope formation, and pathogenesis. To enable these activities, E is likely to be capable of changing its conformation depending on environmental cues. To investigate this issue, here we characterised the structural properties of the C-terminal domain of E (E-CTD), which has been reported to interact with host cell membranes. We first studied the conformation of the E-CTD in solution, finding characteristic features of a disordered protein. By contrast, in the presence of large unilamellar vesicles and micelles, which mimic cell membranes, the E-CTD was observed to become structured. The E-CTD was also found to display conformational changes with osmolytes. Furthermore, prolonged incubation of the E-CTD under physiological conditions resulted in amyloid-like fibril formation. Taken together, these findings indicate that the E-CTD can change its conformation depending on its environment, ranging from a disordered state, to a membrane-bound folded state, and an amyloid state. Our results thus provide insight into the structural basis of the role of E in the viral infection process. HighlightsO_LIThe E-CTD of SARS-CoV-2 is intrinsically disordered in solution C_LIO_LIThe E-CTD folds into an ordered structure in presence of membrane mimetics C_LIO_LIThe E-CTD displays conformational changes in the presence of osmolytes C_LIO_LIProlonged incubation of the E-CTD leads to its self-assembly into amyloid-like fibrils C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/424646v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a2d39aorg.highwire.dtl.DTLVardef@1f2581aorg.highwire.dtl.DTLVardef@17639eaorg.highwire.dtl.DTLVardef@d43ba1_HPS_FORMAT_FIGEXP M_FIG Structural heterogeneity of the E-CTD. The E-CTD shows a disordered secondary structure in an aqueous solution and converts into an ordered structure in the presence of membrane mimetics (neutral and negative lipids) and natural osmolytes (TMAO). Incubation at physiological condition shows typical amyloid-like fibrils. The yellow-colored structure represents a predicted structure of the E-CTD by PEP-FOLD. C_FIG

biophysics↗

bioRxiv2020

Translation-associated mutational U-pressure in the first ORF of SARS-CoV-2 and other coronaviruses

Within four months of the ongoing COVID-19 pandemic caused by SARS-CoV-2, more than 250 nucleotide mutations have been detected in the ORF1 of the virus isolated from different parts of the globe. These observations open up an obvious question about the rate and direction of mutational pressure for further vaccine and therapeutics designing. In this study, we did a comparative analysis of ORF1a and ORF1b by using the first isolate (Wuhan strain) as the parent sequence. We observed that most of the nucleotide mutations are C to U transitions. The rate of synonymous C to U transitions is significantly higher than the rate of nonsynonymous ones, indicating negative selection on amino acid substitutions. Further, trends in nucleotide usage bias have been investigated in 49 coronaviruses species. A strong bias in nucleotide usage in fourfold degenerated sites towards uracil residues is seen in ORF1 of all the studied coronaviruses. A more substantial mutational U pressure is observed in ORF1a than in ORF1b owing to the translation of ORF1ab via programmed ribosomal frameshifting. Unlike other nucleotide mutations, mutational U pressure caused by cytosine deamination, mostly occurring in the RNA-plus strand, cannot be corrected by the proof-reading machinery of coronaviruses. The knowledge generated on the direction of mutational pressure during translation of viral RNA-plus strands has implications for vaccine and nucleoside analogue development for treating covid-19 and other coronavirus infections.

bioinformatics↗