Assessing frailty in older Indian patients before cancer treatment: Comparative analysis of three scales and their implications for overall survival.

INTRODUCTION: Frailty, characterized by ageing-related vulnerability, influences outcomes in older adults. Our study aimed to investigate the relationship between frailty and clinical outcomes in older Indian patients with cancer. MATERIALS AND METHODS: Our observational single-centre study, conducted at Tata Memorial Hospital from February 2020 to July 2022, enrolled participants aged 60 years and above with cancer. Frailty was assessed using the Clinical Frailty Scale (CFS), G8, and Vulnerable Elders Survey (VES)-13. The primary objective was to explore the correlation between baseline frailty and overall survival. Statistical analyses include Kaplan-Meier, Cox proportional hazards, and Harrell's C test. RESULTS: A total of 1,177 patients (median age 68, 76.9% male) were evaluated in the geriatric oncology clinic. Common malignancies included lung (40.0%), gastrointestinal (35.8%), urological (11.9%), and head and neck (9.0%), with 56.5% having metastatic disease. Using CFS, G8, and VES-13 scales, 28.5%, 86.4%, and 38.0% were identified as frail, respectively. Median follow-up was 11.6 months, with 43.3% deaths. Patients fit on CFS (CFS 1-2) had a median survival of 28.02 months, pre-frail (CFS 3-4) 13.24 months, and frail (CFS ≥5) 7.79 months (p < 0.001). Abnormal G8 (≤14) and VES-13 (≥3) were associated with significantly lower median survival (p < 0.001). Multivariate analysis confirmed CFS's predictive power for mortality (p < 0.001), with hazard ratios [HRs] for pre-frail at 1.61(95% confidence interval [CI] 1.25 to 2.06) and frail at 2.31 (95%CI 1.74 to 3.05). G8 ≤ 14 had HR 2.00 (95%CI 1.42 to 2.83), and abnormal VES-13 had HR 1.36 (95%CI 1.11-1.67). In the likelihood ratio test, CFS significantly improved the model fit (p < 0.001). Harrell's C index for survival prediction was 0.62 for CFS, 0.54 for G8, and 0.58 for VES-13. DISCUSSION: In conclusion, our study highlights varying frailty prevalence and prognostic implications in older Indian patients with cancer, emphasizing the need for personalized care in oncology for this aging population. We would recommend using CFS as a tool to screen for frailty for older Indian patients with cancer.

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.

Structural basis for specific RNA recognition by the alternative splicing factor RBM5.

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains to cis-regulatory RNA elements. A structure of the RRM1-ZnF1 region in complex with RNA shows how the tandem domains cooperate to sandwich target RNA and specifically recognize a GG dinucleotide in a non-canonical fashion. While the RRM1-ZnF1 domains act as a single structural module, RRM2 is connected by a flexible linker and tumbles independently. However, all three domains participate in RNA binding and adopt a closed architecture upon RNA binding. Our data highlight how cooperativity and conformational modularity of multiple RNA binding domains enable the recognition of distinct RNA motifs, thereby contributing to the regulation of alternative splicing. Remarkably, we observe surprising differences in coupling of the RNA binding domains between the closely related homologs RBM5 and RBM10.

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein.

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.

Scalable synthesis and structural characterization of reversible KLK6 inhibitors.

Scalable asymmetric syntheses of two kallikrein-related protease 6 (KLK6) inhibitors are reported. The inhibitors are assembled by linking enantiomerically enriched fragments via amide bond formation, followed by conversion of a cyano group to an amidine. One fragment, an amine, was prepared using the Ellman auxiliary, and a lack of clarity in the literature regarding the stereochemical outcome of this reaction was solved via X-ray crystallographic analysis of two derivatives. Complexes of the inhibitors bound to human KLK6 were solved by X-ray crystallography, revealing the binding poses.

Correction: Scalable synthesis and structural characterization of reversible KLK6 inhibitors.

[This corrects the article DOI: 10.1039/D2RA04670A.].

A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.

Structure and dynamics of the quaternary hunchback mRNA translation repression complex.

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

Divergent evolution toward sex chromosome-specific gene regulation in Drosophila.

The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.

Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex.

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.

Transcriptional regulation of the Nε -fructoselysine metabolism in Escherichia coli by global and substrate-specific cues.

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products, such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. While the degradation route for fructoselysine is well described, regulation of the corresponding pathway genes frlABCD remained poorly understood. Here, we used bioinformatics combined with molecular and biochemical analyses and show that fructoselysine metabolism in E. coli is tightly controlled at the transcriptional level. The global regulator CRP (CAP) as well as the alternative sigma factor σ32 (RpoH) contribute to promoter activation at high cAMP-levels and inside warm-blooded hosts, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific repressor. Our study provides profound evidence that the interplay of global and substrate-specific regulation is a perfect adaptation strategy to efficiently utilize unusual substrates within the human gut environment.

Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly.

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.

Pseudo-RNA-Binding Domains Mediate RNA Structure Specificity in Upstream of N-Ras.

RNA-binding proteins (RBPs) commonly feature multiple RNA-binding domains (RBDs), which provide these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability define the specificity of their interactions with RNA. However, how multiple RBDs recognize their cognate single-stranded RNA (ssRNA) sequences in concert remains poorly understood. Here, we use Upstream of N-Ras (Unr) as a model system to address this question. Although reported to contain five ssRNA-binding cold-shock domains (CSDs), we demonstrate that Unr includes an additional four CSDs that do not bind RNA (pseudo-RBDs) but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Disrupting the interactions between canonical and non-canonical CSDs impacts RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies reveal a new paradigm in protein-RNA recognition, where interactions between RBDs and pseudo-RBDs select RNA tertiary structures, influence RNP assembly, and define target specificity.

Integrative Structural Biology of Protein-RNA Complexes.

Ribonucleoprotein complexes (RNPs) are central to all processes in the cell. One of the prerequisites to understand how RNPs work is to determine their high-resolution structures. With the recent revolution in cryoelectron microscopy this task has become easier for large RNP machines, such as ribosomes, spliceosomes, and polymerases. However, the transient and highly dynamic nature of many RNPs makes structure determination a challenging task. Thus, an integrative structural and molecular biology approach is required, tackling three key challenges: (1) identification of cognate RNA sequences; (2) collection of structural data by conducting X-ray crystallography, NMR, electron microscopy, small-angle scattering (SAS), and other experiments; and (3) the creation of structural models that integrates all experimental restraints. Given the breadth of expertise required, this review presents an overview of available methods and successful examples with the goal to provide readers with a selection of promising options for structure determination of RNPs.

Switching the Post-translational Modification of Translation Elongation Factor EF-P.

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands ß3/ß4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli. Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF-Ps from these two model organisms we demonstrate the importance of the ß3/ß4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation.

Selective Inhibitors of FKBP51 Employ Conformational Selection of Dynamic Invisible States.

The recently discovered SAFit class of inhibitors against the Hsp90 co-chaperone FKBP51 show greater than 10 000-fold selectivity over its closely related paralogue FKBP52. However, the mechanism underlying this selectivity remained unknown. By combining NMR spectroscopy, biophysical and computational methods with mutational analysis, we show that the SAFit molecules bind to a transient pocket in FKBP51. This represents a weakly populated conformation resembling the inhibitor-bound state of FKBP51, suggesting conformational selection rather than induced fit as the major binding mechanism. The inhibitor-bound conformation of FKBP51 is stabilized by an allosteric network of residues located away from the inhibitor-binding site. These residues stabilize the Phe67 side chain in a dynamic outward conformation and are distinct in FKBP52, thus rationalizing the basis for the selectivity of SAFit inhibitors. Our results represent a paradigm for the selective inhibition of transient binding pockets.

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless.

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

Mechanism of Mg2+-Accompanied Product Release in Sugar Nucleotidyltransferases.

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg2+ ions. While studying this reaction using X-ray crystallography, we captured snapshots of the pyrophosphate (product) as it exits along a pocket. Surprisingly, one of the active site Mg2+ ions remains coordinated to the exiting pyrophosphate. This hints at the participation of Mg2+ in the process of product release, besides its role in catalyzing nucleotidyl transfer. These observations are further supported by enhanced sampling molecular dynamics simulations. Free energy computations suggest that the product release is likely to be rate limiting in SNTs, and the origin of the high free energy barrier for product release could be traced back to the "slow" conformational change of an Arg residue at the exit end of the pocket. These results establish a dual role for Mg2+, and propose a general mechanism of product release during the nucleotidyl transfer by SNTs.

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P.

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-ß-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.