Recent studies have uncovered various physiological functions of CDK5 in many nonneuronal tissues. Upregulation of CDK5 and/or its activator p35 in neurons promotes healthy neuronal functions, but their overexpression in nonneuronal tissues is causally linked to cancer of many origins. This review focuses on the molecular mechanisms by which CDK5 recruits diverse tissue-specific substrates to elicit distinct phenotypes in sixteen different human cancers. The emerging theme suggests that CDK5’s role as an oncogene or anti-oncogene depends upon its subcellular localization. CDK5 mostly acts as an oncogene, but in gastric cancer, it is a tumor suppressor due to its unique nuclear localization. This indicates that CDK5’s access to certain nuclear substrates converts it into an anti-oncogenic kinase. While acting as a bonafide oncogene, CDK5 also activates a few cancer-suppressive pathways in some cancers, presumably due to the mislocalization of nuclear substrates in the cytoplasm. Therefore, directing CDK5 to the nucleus or exporting tumor-suppressive nuclear substrates to the cytoplasm may be promising approaches to combat CDK5-induced oncogenicity, analogous to neurotoxicity triggered by nuclear CDK5. Furthermore, while p35 overexpression is oncogenic, hyperactivation of CDK5 by inducing p25 formation results in apoptosis, which could be exploited to selectively kill cancer cells by dialing up CDK5 activity, instead of inhibiting it. CDK5 thus acts as a molecular rheostat, with different activity levels eliciting distinct functional outcomes. Finally, as CDK5’s role is defined by its substrates, targeting them individually or in conjunction with CDK5 should create potentially valuable new clinical opportunities. Cyclin-dependent kinases (CDKs) are a family of proline-directed serine/threonine kinases that require binding to cyclins for enzymatic activation. They phosphorylate SP/TP sites with a preference for basic residues on either side , 2. AlthImportantly, recent studies have uncovered that CDK5 and p35 are expressed in the vast majority of nonneuronal tissues, where they regulate various physiological functions, including hematopoietic cell differentiation, immune response, lymphatic valve formation, myogenesis, melanogenesis, insulin levels, cell migration, wound healing, invasion, survival, and angiogenesis. CDK5 deregulation is causatively linked to several diseases, including cancer, senescence, diabetes, immune dysfunction and inflammation . NeverthThe most predominant CDK5 activators are CDK5R1 and CDK5R2 , which were initially believed to be CNS specific; however, later studies confirmed widespread expression of p35 in various tissues under both physiological and pathological conditions. P39, an isoform of p35 with 57% amino acid homology, shows more restricted expression in extraneuronal tissues . In addiIn neuronal cells, CDK5 is physiologically activated by transcriptional upregulation of p35 or p39 in response to many factors, including neurotropic factors, cytokines and growth factors. Under neurotoxic conditions, such as β-amyloid, excitotoxicity, oxidative stress, and calcium deregulation, both p35 and p39 are cleaved by calpain to generate p25 and p29, respectively, which have much longer half-lives due to their resistance to ubiquitin-mediated proteolysis , 19. In Cyclin I (CCNI) is another CDK5-specific activator that binds CDK5 in terminally differentiated cells such as neurons and podocytes . BindingCDK5 expression levels were found to be upregulated by TP53-induced glycolysis regulatory phosphatase (TIGAR) to promote DNA damage repair . RecentlSimilarly, CDK5 possesses its own set of negative regulators and is inhibited by cyclin D1, cyclin E, and GSTP1 , 32, 33.CDK5 is not known to be mutated in cancer. However, unlike neurodegenerative diseases, where CDK5 deregulation is predominantly believed to be due to p25 formation, in cancers, it is mainly due to CDK5 and/or p35 (and sometimes p39) overexpression/amplification, although in medullary thyroid carcinoma (MTC), high p25 levels have been reported  Table 11. AccordImportantly, in gastric cancer, CDK5 levels are much lower than those in normal gastric tissues, which correlates with decreased patient survival and metastases , 41. ThiCDK5 and p35 are highly expressed in breast cancer tissues and positively correlate with tumor progression and poor prognosis , 43 Tab. The mecCDK5 also promotes chemotaxis in breast cancer cells by phosphorylating Talin’s head domain. Calpain cleaves talin, creating a globular head domain and a C-terminal rod domain. The talin head domain binds to Smurf1, an E3 ubiquitin ligase, and is degraded Fig. . The talCDK5 likewise promotes tumorigenesis by inhibiting cell death pathways. Loss of CDK5 in breast cancer cells opens up the mitochondrial permeability transition pore (mPTP), initiating increased mitochondrial depolarization and higher ROS levels, with subsequent activation of caspases and cell death Fig. B. This iSimilar to breast cancer cells, prostate cancer cells express high levels of CDK5 and p35 Table  50, 51], 5150, 5727-STAT3 significantly correlated with Gleason score and CDK5, p35 and AR levels . AsInterestingly, in pancreatic cancer, Ral guanine nucleotide-exchange factors  have emerged as the key effectors of the K-Ras pathway instead of the B-Raf, extracellular signal-regulated kinase (ERK) or MAPK pathways. As Ral GEFs activate RalA and RalB small G proteins, Lim et al. demonstrated that RalA is critical for Ras-driven tumor initiation and that RalB is crucial for Ras-driven tumor metastasis . As CDK5In pancreatic cancer, active CDK5 phosphorylates enhancer of zeste homolog 2 (EZH2), which triggers its degradation via FBW7, a component of ubiquitin ligase , which synthesize calcitonin. MTC accounts for ~ 1–2% of all thyroid cancers and can be sporadic (75% MTC cases) or hereditary (25%). CDK5 and its activators are present in normal human thyroid tissues but overexpressed in hereditary and sporadic MTC clinical specimens Table A 34]..34].FOS and JUNB. Accordingly, phospho-dead STAT3 (S727A) prevented p35-induced human TT and mouse MTC-M cell proliferation  that bind with the nuclear export mediator CRM-1 and result in cytoplasmic residence of CDK5 . Cao et Lu et al. further revealed that downregulation of CDK5 promotes metastasis in gastric cancer cells, which depends on its interaction with PP2A, a serine/threonine phosphatase ; howeverCDK5 and p35 are broadly expressed in human colorectal cancer (CRC) cell lines and human CRC tissues compared to paired normal tissues and cell lines Table  66, 67], 6766, 6Gliomas or GBM are the most common and lethal malignant brain cancer with high recurrence rates. CDK5 and p35 are overexpressed in gliomas compared to normal brain tissues Table  68, 69], 6968, 6Multiple studies have shown that CDK5 increases proliferation  and invaGlioblastoma cell proliferation was also dependent on collapsin response mediator protein 2 (CRMP2) phosphorylation. CDK5 phosphorylates CRMP2 at S522, triggering nuclear translocation and causing cell proliferation Table . NeurofiCDK5 also regulates glioma tumorigenicity by phosphorylating Tripartite motif containing 59 (TRIM59), a ubiquitin ligase. Upon EGF stimulation, CDK5 phosphorylates TRIM59 at S308 and promotes its nuclear translocation via Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (PIN1). PIN1 triggers the cis-trans isomerization of TRIM59, which causes it to bind importin α5, resulting in its nuclear translocation. Nuclear TRIM59 then promotes ubiquitination and degradation of the tumor-suppressive histone variant macroH2A1, causing STAT3 activation and tumorigenicity Fig.  72]. Ad. Ad72]. GBMs preferentially use acetyl-CoA to fuel tumor growth. As a result, GBMs upregulate O-GlcNAc transferase (OGT), which upregulates acetate conversion to acetyl-CoA and lipids via CDK5 and Acyl-CoA Synthetase Short Chain Family Member 2 (ACSS2). OGT-mediated O-GlcNAcylation increases CDK5 activity, which phosphorylates ACSS2 at S267, inhibiting its degradation and promoting the synthesis of acetyl-CoA from acetate . ACSS2 pGlioblastoma exhibits striking tumor heterogeneity leading to cellular hierarchies with self-renewing glioblastoma stem cells (GSCs), also known as brain tumor initiating cells (BTICs), at the apex. GSCs or BITCs are critical drivers of tumor initiation, progression and therapeutic resistance. Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) promotes differentiation and inhibits stemness acquisition of GSCs by inhibiting CDK5. Although the exact mechanism remains unknown, DYRK1A inhibition or depletion upregulates the CDK5 activators p35 and p39. This causes CDK5 activation, which in turn enhances SOX2 expression, leading to stemness . Xie et Interestingly, Catalina et al. observed that a small subset of GBM tumor sections with high CDK5 and p35/p25 levels exhibited apoptotic features. This prompted them to irradiate GBM cells, which did not alter CDK5 levels, but significantly increased p25 levels. Increased p25 formation strongly correlated with enhanced apoptosis , indicatHCC is the third leading cause of cancer-related deaths worldwide. Using a large cohort of samples, Zhang et al. showed that CDK5 expression levels were significantly higher in HCC tissues than in normal, cirrhotic, and adjacent noncancerous liver tissues. Furthermore, CDK5 upregulation revealed a positive correlation with metastasis, vascular invasion, differentiation, advanced clinical stages, decreased survival and greater tumor recurrence Table  77, 78], 7877, 7Lnc-ATG9B-4, a long noncoding RNA, is overexpressed in hepatic cancer and is strongly associated with tumor size, TNM stage, portal vein tumor thrombus, metastasis and poor prognosis. Li et al. showed that CDK5 mRNA levels are upregulated by lnc-ATG9B-4 in hepatic cancer, resulting in aggravated oncogenic phenotypes   ~ 15%) and non-small cell lung cancer (NSCLC) (remaining 85%). SCLCs are significantly more aggressive than NSCLCs. CDK5 regulates the prognosis of both SCLCs and NSCLCs, and its overexpression is associated with several clinicopathological factors and poor patient survival . TTN-AS1 is upregulated in LUAD tissues and associated with TNM stage, lymph node metastasis and poor postoperative prognosis. TTN-AS1 depletion significantly inhibits the proliferation and invasion ability of LUAD cells. TTN-AS1 functions as a competing endogenous RNA by sponging miR-142-5p to upregulate CDK5 in LUAD  Fig. 6AA6A. miR-Several downstream targets of CDK5 have been identified in lung cancer. In NSCLC, CDK5 triggers cyclase-associated protein 1 (CAP1) phosphorylation at S308 and S310 (human CAP1 numbering), resulting in actin remodeling . CAP1 phProgrammed cell death ligand 1 (PD-L1) is an immune checkpoint protein that is often overexpressed on tumor cells, including lung cancer cells. It binds to programmed cell death protein 1 (PD-1) on T cells and inhibits CD8 T-cell activity, allowing cancer cells to evade immune elimination. De et al. reported that high levels of FBXO22 (a ubiquitin E3 ligase) sensitize NSCLC cells to cisplatin and ionizing radiation (IR) by degrading PD-L1 . IR induParadoxically, CDK5 can also promote the tumor-suppressing functions of Deleted in Liver Cancer 1 (DLC1) in lung cancer . DLC1, aMalignant melanoma is the deadliest form of skin cancer, as it is clinically very aggressive and metastatic. CDK5 and its activator p35 are highly expressed in human melanoma cells and tissues Table  101, 10. ActivatCDK5 was found to regulate melanoma cell invasiveness by directly phosphorylating vimentin . VimentiA central role of CDK5 in antitumor immunity was recently reported by Dorand et al.  in subcuHigh CDK5 expression is associated with shorter progression-free survival in ovarian cancer Table  105]. F. F105]. Medulloblastoma is the most common pediatric brain malignancy and frequently arises in the cerebellum. Medulloblastoma cell lines and clinical specimens express CDK5, p35 and p39 Table  103]. D. D103]. Aberrant overexpression of CDK5 significantly induces tumor cell motility and EMT in HNSCC Table  108]. S. S108]. So et al. observed that CDK5, p35 and Cyclin I mRNA levels are increased in acute myeloid leukemia (AML) patients in The Cancer Genome Atlas dataset Table  109]. I. I109]. CDK5 promotes growth and survival by phosphorylating BH3-only protein NOXA in hematopoietic cells. NOXA is constitutively expressed in proliferating cells of the hematopoietic lineage. NOXA interacts almost exclusively with the prosurvival Bcl-2 protein Mcl-1L, inactivating its survival function and thereby promoting apoptosis  Fig. 8BB8B. HoweSo et al. demonstrated that CDK5 or CCNI loss sensitized cells to mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) inhibition, while p35 knockdown did not, indicating that CDK5 likely partners with CCNI in AML . Neverth2/M checkpoint to prevent passage into mitosis upon DNA damage [Multiple myeloma (MM) is an incurable plasma cell malignancy of bone marrow. Increased expression of CDK5 is observed in both primary MM cells and MM cell lines compared to normal bone marrow mononuclear cells (BMMCs) Table  111, 11. High exA damage , a lack Eph receptor A4 (EphA4) is overexpressed in several tumors and promotes the proliferation of multiple myeloma cells. EphA4 binds CDK5 and increases its expression, leading to enhanced AKT activation and cell adhesion-mediated drug resistance in multiple myeloma .CDK5 has emerged as a critical player in both cancer and neurodegenerative diseases. Nevertheless, some critical differences govern CDK5 deregulation modes in these disparate diseases. While CDK5 and/or p35 upregulation in neurons promotes healthy neuronal functions, their upregulation in cancer promotes highly aggressive phenotypes Fig. C. SimilaIn neurodegenerative diseases, p25 formation is crucial for CDK5 deregulation due to its long half-life and by mislocalizing active CDK5 to the cytoplasm and nucleus. In contrast, p25’s contribution to cancer progression is less understood. In pancreatic cancer, K-Ras leads to p25 formation through an unknown mechanism. However, as the vast majority of pancreatic tumors overexpress CDK5, p35 and p39, the contribution of p25 to disease pathogenesis remains unclear. Bibb et al. created an inducible p25 MTC mouse model in which proliferation was triggered upon p25 expression in C-cells and halted by interrupting p25 overexpression . As bothIn neurons, an initial increase in p25 levels promotes synaptic plasticity; however, an increase beyond a certain threshold leads to neurodegeneration. We believe a similar mechanism exists in cancer, where a moderate increase in p25 may promote oncogenicity, but high levels of p25 are fatal. A few studies have indeed shown that hyperactivation of CDK5 by inducing p25 formation causes apoptosis in cancer , 117. InAn increase in CDK5 levels or activity is frequently associated with highly aggressive oncogenic phenotypes and a poor prognosis. However, in these cancers, CDK5 also inhibits oncogenic pathways through a few substrates. CDK5 activates tumor-suppressive pathways in pancreatic cancer via EZH2 degradation and in NSCLC by DLC1 activation. As DLC1 is often downregulated in lung cancer, CDK5-mediated DLC1 activation may have a limited impact. Likewise, EZH2 is predominantly nuclear in lung cancer. Therefore, CDK5-triggered degradation of cytoplasmic EZH2 may also have little effect. Uniquely in gastric cancer, CDK5 acts as a tumor suppressor. This has been attributed to its nuclear localization, allowing it access to distinct substrates. Although CDK5-EZH2 crosstalk has not been analyzed in gastric cancer, it is possible that CDK5 degrades nuclear EZH2 in gastric cancer, triggering tumor-suppressive pathways. Finally, as both EZH2 and CDK5 have independently been shown to be critical for NEPC progression, it is likely that they reside in different compartments where each promotes specific oncogenic pathways. Future studies are needed to uncover their potential crosstalk in NEPC.Most importantly, analysis of CDK5’s substrates and pathways in sixteen different cancers suggests that CDK5’s behavior as an oncogene or an anti-oncogene depends upon its subcellular localization Table . Based oFinally, more than two dozen direct substrates of CDK5 have been identified in cancer to date Table . Some of


As sequence and structural databases grow along with powerful analysis tools, the prevalence and diversity of pseudoenzymes have become increasingly evident. Pseudoenzymes are present across the tree of life in a large number of enzyme families. Pseudoenzymes are defined as proteins that lack conserved catalytic motifs based on sequence analysis. However, some pseudoenzymes may have migrated amino acids necessary for catalysis, allowing them to catalyze enzymatic reactions. Furthermore, pseudoenzymes retain several non-enzymatic functions such as allosteric regulation, signal integration, scaffolding, and competitive inhibition. In this review, we provide examples of each mode of action using the pseudokinase, pseudophosphatase, and pseudo ADP-ribosyltransferase families. We highlight the methodologies that facilitate the biochemical and functional characterization of pseudoenzymes to encourage further investigation in this burgeoning field. Pseudoenzymes are proteins that lack conserved catalytic amino acids when compared with their catalytically active counterparts. Once considered inactive or dead, these ‘zombie enzymes' carry out a large variety of non-enzymatic functions across the tree of life, in addition to occasional enzymatic functions . The abuIdentification of pseudoenzymes relies on computational methods to detect proteins that diverge at highly conserved catalytic residues based on sequence or structural alignments. A major limitation of such computational methods is the fact that they rely on our current understanding of the catalytic mechanisms. One of the most well-studied signaling families is the kinase superfamily, consisting of protein and small molecule kinases. In 1988, Hanks et al.  generatePlasmodium falciparum and Giardia lamblia consist of pseudokinases [Halorientalis regularis are pseudokinases, highlighting the diversity in the size of pseudokinomes [The kinome comprises of all the protein kinases identified in a genome . In eukaokinases . Bacteriokinomes .The kinome is divided into groups based on sequence, structural, functional, and evolutionary similarity ,9. The pin vitro [+K+2Cl− cotransporter (NKCC1) [One example of a pseudokinase with ties to pathophysiology is with no lysine (WNK1) kinase, which lacks the conserved lysine in the VAIK motif. Mutations in WNK1 are associated with autosomal dominant pseudohypoaldosteronism characterized by hyperkalemia and hypertension . WNK1 wain vitro . Phospho (NKCC1) ,19. The  (NKCC1) .Danio rerio POMK illustrates that the migrated DLD and MCD motifs serve the roles of the canonical DFG and HRD motifs, respectively [Similarly, the protein-O mannose kinase (POMK), also known as SGK196, is an atypical kinase once predicted to be catalytically inactive . POMK laectively . Consistectively .Pseudomonas syringae SelO revealed that ATP binds in an inverted orientation in the active site when compared with canonical kinases [Excitingly, recent studies have demonstrated that certain pseudokinases may catalyze alternative transferase activities. The pseudokinase SelO is conserved from bacteria to humans, suggesting an important role in biology ,28. This kinases . Structu kinases . Given t kinases . Notably kinases . These a kinases . As demo kinases . In line kinases .in vitro activity [In vitro and in cell co-expression studies demonstrate that the expression of Fam20A increases the activity of Fam20C. Structural analysis revealed the mechanisms by which Fam20A increases the catalytic efficiency of Fam20C through the formation of Fam20A–Fam20C heterodimer [Pseudokinases with enzymatic activity could arguably be termed atypical kinases, although many have significantly decreased enzymatic activity in comparison with canonical kinases with conserved architecture ,24. Baseactivity . One sucactivity . An examactivity . Fam20A activity . Mutatioactivity . Howeveractivity ,46. In verodimer ,47. Fam2in cis or in trans models of regulation [in cis [in trans inhibition of the JH1 kinase domain of one protein molecule by the JH2 pseudokinase domain of another protein molecule [in cis with the kinase domain and in trans with another pseudokinase domain [in cis with the kinase domain, but also via dimer formation in trans with the opposing pseudokinase domain [In contrast with the Fam20A and Fam20C dimer, the JAK pseudokinase domain acts as an allosteric inhibitor of JAK activity. The receptor-bound JAKs contain both a kinase (JH1) and a pseudokinase (JH2) domain in the same polypeptide as a result of domain duplication . Removalgulation . The cry [in cis . In contmolecule . The firmolecule . Glassmamolecule  utilizede domain . Oncogene domain . These re domain ,54. Moree domain . Interese domain .In addition to serving as allosteric regulators, pseudokinases may also act as molecular switches in signaling pathways . The strStructural analysis of the pseudokinase domains of rat, mouse, horse, and human orthologues of MLKL depict conformational variations that contribute to the different mechanisms of MLKL activation in mice and humans . In partA common theme in several proteins harboring the pseudokinase domain is the presence of additional accessory domains, as seen with MLKL. Another pseudokinase family that harbors accessory domains is the family of Tribbles pseudokinases , which lacks the aspartate in the DFG motif and harbors an E3 ligase binding motif in the C terminus ,68. The These studies exemplify the biochemical and functional versatility of the pseudokinase superfamily . Additio5R catalytic motif [Chen et al.  identifiic motif .5R motif [in vitro pulldown assays demonstrate that STYX interacts with ERK2, predominantly in the nucleus. The ability to bind the same ligand as an active counterpart can serve multiple purposes, with such pseudoenzymes potentially restraining ligands in specific locations or serving as competitive inhibitors [Mutation of a single amino acid can revert some CC1 pseudophosphatases to active enzymes ,80,81. O5R motif . Bindinghibitors . STYX sehibitors .As demonstrated with STYX and other pseudophosphatases, the substrate-trapping mechanism of pseudophosphatases can be harnessed to identify binding partners ,84. Simi+ to substrates to form a mono-ADP ribose or poly-ADP ribose modification [Another family of emerging pseudoenzymes is the ADP-ribosyltransferase (ART) like proteins lacking the classical active site. ARTs catalyze the transfer of ADP ribose from NADfication . Membersfication . Amino afication . Using sfication  identifi+ mimetic nor catalyze poly-ADP-ribosylation in vitro [The transgene activation suppressor protein (TASOR) was recently identified to harbor a pseudoART domain that forms the human silencing hub (HUSH) complex along with M-phase phosphoprotein 8 (MPP8) and Periphilin . The HUSin vitro . HoweverMagnaporthe oryzae, provided a numerical estimate of the degree of divergence from an estimated consensus sequence for each protein and mapped the most conserved residues [The ART family provides clues about the evolutionary origin of pseudoenzymes, suggesting that pseudoenzymes may have evolved from catalytically active ancestors. Shannon's entropy analysis of ARTs in the secreted proteins of a fungal phytopathogen, residues . ARTs prresidues ,96. Thisresidues ,97. Whenresidues ,99. The The previous examples of pseudoenzymes have functions related to that of their catalytic counterparts, either by binding the same ligands or regulating their counterparts. However, over 300 proteins across the domains of life display moonlighting activity characterized by multiple distinct functions carried out by the same protein . CrystalPseudoenzymes arising from a catalytically active enzyme retain the overall structural fold to function. As proposed by Pils et al., the loss of catalytic amino acids in a pseudoenzyme may reduce selective pressure to conserve catalysis, and thus support alternative conformations and uses for the active site ,95. ThisTrypanosoma brucei protein arginine methyltransferase pseudoenzyme (TbPMRT1 PRO) forms a complex with its enzymatic counterpart, TbPRMT1 ENZ. Like Fam20A, TbPMRT1 PRO forms a complex with TbPRMT1 ENZ to increase its partner's catalytic activity [The activity . TbPMRT1activity . Size exactivity . StructuM. oryzae, used TrRosetta structural algorithms to explore, among other things, the evolution of pseudoADP-ribosyl transferases by comparing their predicted structures to active counterparts. Notably, this survey also revealed several proteins with analogous structures but divergent sequences, suggesting that unrelated sequences might result in similar functions [The examples above demonstrate the utility of bioinformatic analysis and structural genomics in guiding experimental validation of pseudoenzyme function . Sequencunctions .in vitro may be artificial and may not reflect its function in cell. Some suggested methods to aid in the study of pseudoenzymes include immunoprecipitation and pulldown assays to identify interaction partners, intact mass analysis to detect post-translational modification, and structural and mutational analysis paired with biochemical assays to understand the molecular basis of activity. Conformational trapping of pseudoenzymes using monobodies or nanobodies is particularly useful to stabilize a single conformation for structural studies. Caution must be exercised when assaying immunoprecipitated pseudoenzymes, as contaminating enzymes may skew the analysis. A technique that is amenable to high-throughput screening is the SYPRO Orange thermal shift assay. Ligand binding increases the thermal stability of some proteins as seen with ATP binding to pseudokinases. Thermal stability of a protein can be quantified as a measure of increasing fluorescence upon protein denaturation and SYPRO orange binding to hydrophobic patches on proteins [We are at an exciting time in history with new and exciting biology on the horizon of pseudoenzymes. Although we understand the functional role of some pseudoenzymes, we lack an understanding of the biochemical mechanisms of action. Experimental validation is limited by the identification of optimal conditions and physiological substrates, necessity for cofactors, and the understanding of catalytic mechanism/biochemical reaction. Weak or vestigial activity of pseudoenzymes proteins . The stuThe pseudokinases are the most extensively studied pseudoenzyme family and a major target for drug discovery ,114. The


Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides’ opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2′-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.The macrocyclic tetrapeptide CJ-15,208 ( The majority of opioid ligands used clinically for the treatment of pain are mu opioid receptor (MOR) agonists, although agonists of the kappa (KOR) and delta (DOR) opioid receptors also produce analgesia. Opioid analgesics remain efficacious therapeutics for moderate to severe pain, but their use is limited by clinical liabilities, including concurrent substance abuse disorders . MOR-selcyclo[Phe-D-Pro-Phe-Trp], d-Trp isomer of CJ-15,208 primarily exhibited KOR antagonism with modest antinociception only at elevated doses CJ-15,208 surprisingly demonstrated mixed MOR-agonist/DOR-antagonist activity, prompting us to hypothesize that it would demonstrate both decreased antinociceptive tolerance in an acute assay and potentially prevent reinstatement of extinguished cocaine-conditioned place preference.In the current study, we explored the effects of substitutions for the tryptophan residues in CJ-15,208 and CJ-15,208 analogs generally increased relative to the parent peptide. The KOR affinity of the D-Bta4 analog was 5-fold higher than [D-Trp]CJ-15,208, while the MOR affinities of the D-Bta4 and D-Nal(2′)4 were 3- to 4-times higher than the parent peptide. All of the analogs exhibited at best low (micromolar) affinity for DOR, similar to the parent peptides.The affinities of the analogs were determined in radioligand equilibrium competition binding assays . The KOR35S]GTPγS assay to assess KOR agonist activity. Consistent with the results for CJ-15,208 and [d-Trp]CJ-15,208 [35S]GTPγS assays.Analogs were also evaluated in a CJ-15,208 was mediated by the MOR and KOR .Contributions of individual opioid receptors to antinociception observed with CJ-15,208 and the analogs of CJ-15,208 and [D-Trp]CJ-15,208 receptor . Except f analog . The Bta = 3.41, p = 0.02, one-way ANOVA, but without specific significance in Dunnett’s post-hoc testing;  = 1.30, p = 0.29, one-way ANOVA;  = 34.3, p < 0.0001, one-way ANOVA with Dunnett’s post-hoc test), substitution of other L-amino acids for Trp abolished this property  = 54.8, p < 0.0001, one-way ANOVA with Dunnett’s post-hoc test;  = 15.4, p < 0.0001, one-way ANOVA with Dunnett’s post-hoc test;  = 4.53, p = 0.004, one-way ANOVA; p = 0.08 or higher, Dunnett’s post hoc test).Following the dissipation of any antinociception, the analogs were evaluated for antagonist activity against the MOR-preferring agonist morphine , the KOR-selective agonist U50,488 , and the DOR-selective agonist SNC-80 4]CJ-15,208 and morphine were tested in a model of acute antinociceptive tolerance CJ-15,208 treatment significantly reduced the ED50 values collected at 8 h vs. 0 h, with a leftward 2.7-fold shift in the dose–response curve of the second dose administered olerance  with repnistered .4]CJ-15,208 was assessed at the peak antinociceptive dose  for its effects on respiration rates and spontaneous locomotor activity over an 80 min period using the Comprehensive Laboratory Animal Monitoring System (CLAMS)  = 2.38, p = 0.01, two-way RM ANOVA with Tukey’s multiple comparison post hoc test; 4]CJ-15,208 did not significantly impair breathing, and in fact statistically increased it briefly from 20 to 40 min compared to vehicle  = 5.19, p < 0.0001, two-way RM ANOVA;  = 4.97, p = 0.03, two-way RM ANOVA) and not [Nal(2′)4]CJ-15,208  = 1.40, p = 0.25, not significant, two-way RM ANOVA).[Nal(2′) (CLAMS) . As expe4]CJ-15,208 was assessed for rewarding or aversive properties using a conditioned place preference (CPP) assay. Following a two-day place conditioning paradigm, mice conditioned with morphine  demonstrated a significant (p = 0.03) place preference for the morphine-paired chamber, whereas mice conditioned with U50,488  demonstrated a significant (p = 0.03) conditioned place avoidance  = 4.89, p = 0.004, two-way ANOVA with Sidak’s multiple comparison post hoc test; 4]CJ-15,208 at either a 30 or 100 nmol, i.c.v. dose demonstrated no significant preference or aversion for their drug-paired chamber 4]CJ-15,208 was tested for its ability to prevent stress- and cocaine-induced reinstatement of extinguished cocaine-seeking behavior  = 45.9, p < 0.0001, one-way RM ANOVA with Tukey’s multiple comparisons post hoc test; p = 0.48 vs. pre-conditioning response, and p < 0.0001 vs. post-conditioning response, Tukey’s post hoc; 4]CJ-15,208  and exposed to forced swim stress (FSS) or a single additional cycle of cocaine place conditioning  = 15.2, p < 0.0001, one-way ANOVA with Tukey’s post hoc; 4]CJ-15,208 dose-dependently prevented stress-induced reinstatement of cocaine-CPP; while a 10 nmol dose proved ineffective  = 25.2, p < 0.0001, one-way ANOVA with Tukey’s post hoc; 4]CJ-15,208  prior to the additional cocaine place conditioning did display a place preference significantly smaller than that of the initial cocaine-CPP , the response did not significantly differ from that of the vehicle-treated, cocaine-place conditioned mice behavior . Followii ratio ≤ 5), except for the D-Bta4 derivative, where the 5-fold increase in KOR affinity increased KOR selectivity over the MOR. Similar to the parent peptides, all of the analogs exhibited micromolar affinity for the DOR. The stereochemistry of the residue in this position also affected the metabolic stability; similar to the parent peptides CJ-15,208 was mediated only by MOR. In contrast, two of the three analogs containing a D-amino acid exhibited minimal (20–30%) antinociception similar to [D-Trp]CJ-15,208; only [D-Nal(2′)4]CJ-15,208 exhibited maximum antinociception, although this was only at high doses.The antinociceptive activity of the analogs paralleled the results for the respective parent peptides. The peptides containing an L-amino acid at this position all produced maximum antinociception following i.c.v. administration, although the potency varied over an 80-fold range. While the antinociception produced by both the Bta4]CJ-15,208 antagonized antinociception produced by the KOR agonist U50,488, in contrast to the KOR antagonism produced by both of the parent peptides. Unexpectedly, [Nal(2′)4]CJ-15,208 significantly antagonized the DOR agonist SNC-80.Only two of the analogs exhibited antagonist activity when tested against standard opioid agonists. Only [D-Bta4]CJ-15,208 for several potential liabilities associated with MOR agonists. Significant tolerance develops to the analgesic effects of MOR agonists such as morphine, requiring higher doses to achieve analgesia. In contrast to morphine, [Nal(2′)4]CJ-15,208 did not produce acute tolerance, and its antinociceptive ED50 actually decreased significantly in animals pretreated with the peptide. Functional interactions between mu- and delta-opioid receptors , as evieference ,7,8, whieference , and DOReference . Extendi opioids . More ex4]CJ-15,208 proved effective at doses consistent with those that decreased the antinociceptive efficacy of SNC-80. Consistent with this interpretation, the DOR-selective antagonist SoRI-9409 reduced yohimbine stress-induced reinstatement of alcohol-seeking behavior in rats [The current results with [Nal(2′)]CJ-15,208 represent the first demonstration that a mixed action MOR agonist/DOR antagonist compound is effective at preventing stress-induced reinstatement of extinguished cocaine-conditioned place preference. Such effects are likely mediated by DOR antagonism, given that   and N,N-diisopropylethylamine  in DMF. After 15 h, additional HATU (0.75 equiv) was added in one portion to the reaction, and additional linear peptide  was added dropwise (1.2 mL/h) as described above . The reaction was stirred for 6 h at room temperature, and then for an additional 24 h at 37 °C. The solvent was evaporated under reduced pressure, and the crude cyclic peptide isolated as previously described CJ-15,208  as a white solid following lyophilization.Following cyclization of the linear peptide  the macrocyclic peptide was purified by flash chromatography using a step gradient of 60 to 100% EtOAc (10% increments) in hexanes, followed by a step gradient of 0 to 3% MeOH (1% increments) in EtOAc to yield pure [Nal(1′)4]CJ-15,208  as a white solid following lyophilization.Following cyclization of the linear peptide , the macrocyclic peptide was purified using a gradient of 50 to 100% EtOAc in hexanes over 20 min to give pure [Nal(2′)4]CJ-15,208  as a white solid following lyophilization.Following cyclization of the linear peptide , the macrocyclic peptide was purified using a gradient of 30 to 100% EtOAc in hexanes over 20 min to give pure [Bta4]CJ-15,208  as a white solid following lyophilization.Following cyclization of the linear peptide , the macrocyclic peptide was purified using a gradient of 40 to 100% EtOAc in hexanes over 20 min to give pure [D-Nal(1′)4]CJ-15,208  as a white solid following lyophilization.Following cyclization of the linear peptide , the macrocyclic peptide was purified using a gradient of 30 to 100% EtOAc in hexanes over 20 min to give pure [D-Nal(2′)2]CJ-15,208 diprenorphine, [3H]enkephalin (DAMGO), and [3H]enkephalin (DPDPE), respectively. Following determination of IC50 values by nonlinear regression using Prism software , Ki values were calculated using the Cheng–Prusoff equation GTPγS binding to membranes from CHO cells stably expressing opioid receptors was determined as previously described CJ-15,208 was instead determined by pretreating mice with a single dose of β-funaltrexamine  or nor-BNI  24 h in advance of administration of a dose of a macrocyclic peptide. Assessment of DOR involvement in the agonist activity of each macrocyclic peptide was performed by pretreating additional wild-type mice with a single dose of naltrindole  20 min in advance of administration of the macrocyclic peptide.The opioid receptor involvement in the agonist activity of the macrocyclic peptides was determined by testing in MOR KO and KOR KO mice as utilized previously , except To determine antagonist activity, mice were pretreated with the macrocyclic peptide 140 min prior to the administration of the MOR-preferring agonist morphine , KOR-selective agonist U50,488 , or DOR-selective agonist SNC-80 ; at this time, the antinociceptive activity of the analogs had dissipated. Antinociception produced by the established opioid agonists was then measured 40 min after their administration. The dose response of KOR or DOR antagonism produced by selected analogs was further determined by evaluating antinociception after administration of one of three additional i.c.v. doses of the analog.50 dose (i.c.v.) of test compound in the morning (time = 0) and a second dose  8 h later. The degree of tolerance was calculated from the shift in ED50 value from the singly to repeatedly treated condition CJ-15,208 , morphine , or vehicle, as indicated, and 5 min later confined to the CLAMS testing chambers. Pressure monitoring within the sealed chambers measured frequency of respiration. Infrared beams located in the floor measured locomotion as number of beam breaks. Respiration and locomotive data were averaged over 20 min periods for 80 min post-injection of the test compound. Data are presented as % vehicle response ± SEM; ambulation, or breaths per minute.Respiration rates (in breaths per minute) and animal locomotive activity (as ambulation) were assessed using the Oxymax/CLAMS system  as described previously ,63. Micep > 0.05, Student’s t-test). For place conditioning, mice were administered 0.9% saline (i.p.) and consistently confined in a randomly assigned outer compartment: half of each group in the right chamber, and half in the left chamber. Four hours later, mice were administered test compound and confined to the opposite compartment for 40 min. To determine if [Nal(2′)4]CJ-15,208  produced CPP or CPA, mice were place conditioned in this way for two days, with a final preference test taken on the fourth day, as this has been shown to produce dependable morphine-CPP and U50,488-induced CPA CJ-15,208  20 min prior to cocaine  for an additional day of place conditioning. On the day following the completion of stress exposure or cocaine place conditioning, mice were tested for place preference to determine possible reinstatement  values and 95% C.I. were determined using individual data points from graded dose–-response curves with Prism 8.0 software . Percent antinociception was used to determine within-group effects and to allow comparison to baseline latency in tail-withdrawal experiments. The statistical significance of differences between ED50 values was determined by evaluation of the ED50 value shift via nonlinear regression modeling with Prism software. Significant differences in behavioral data were analyzed by ANOVA (one-way or two-way with repeated measures (RM), as appropriate). Significant results were further analyzed with Sidak’s, Tukey’s, or Dunnett’s multiple comparison post hoc tests, as appropriate. Data for conditioned place preference experiments were analyzed by one-way or two-way RM ANOVA, with analyses examining the main effect of conditioned place preference phase  and the interaction of drug pretreatment and/or exposure. All data are presented as mean ± SEM, with significance set at p < 0.05.All dose–response lines were analyzed by regression, and ED4]CJ-15,208 retaining KOR antagonism. Unexpectedly, the novel macrocyclic tetrapeptide [Nal(2′)4]CJ-15,208 demonstrated bifunctional MOR agonism and DOR-selective antagonism, producing antinociception in the mouse 55 °C warm-water tail-withdrawal assay without the clinical liabilities associated with standard MOR agonists, namely tolerance, respiratory depression, or psychostimulation, and lacked rewarding properties in a conditioned place preference paradigm. Notably, [Nal(2′)4]CJ-15,208 prevented stress-induced reinstatement of extinguished cocaine-conditioned place preference in a dose-dependent manner. Collectively, these findings suggest that [Nal(2′)4]CJ-15,208 along with other multifunctional opioid agonist/antagonist compounds are promising candidates for therapeutic development for pain management and/or substance abuse.KOR antagonism was sensitive to substitution of the L- or D-Trp residue, with only [D-Bta


Acinetobacter baumannii is a major cause of hospital-acquired opportunistic infections. The increasing spread of pan-drug resistant strains makes A. baumannii top-ranking among the ESKAPE pathogens for which novel routes of treatment are urgently needed. Comparative genomics approaches have successfully identified genetic changes coinciding with the emergence of pathogenicity in Acinetobacter. Genes that are prevalent both in pathogenic and a-pathogenic Acinetobacter species were not considered ignoring that virulence factors may emerge by the modification of evolutionarily old and widespread proteins.The Gram-negative bacterial pathogen Acinetobacter. In most pathogenic Acinetobacter isolates, ComC displays a von Willebrand Factor type A domain harboring a finger-like protrusion, and we provide experimental evidence that this finger conveys virulence-related functions in A. baumannii. All three genes are part of an evolutionary cassette, which has been replaced at least twice during A. baumannii diversification. The resulting strain-specific differences in T4aP layout suggests differences in the way how individual strains interact with their host. Our study underpins the hypothesis that A. baumannii uses T4aP for host infection as it was shown previously for other pathogens. It also indicates that many more functional complexes may exist whose precise functions have been adjusted by modifying individual components on the domain level.Here, we increased the resolution of comparative genomics analyses to also include lineage-specific changes in protein feature architectures. Using type IVa pili (T4aP) as an example, we show that three pilus components, among them the pilus tip adhesin ComC, vary in their Pfam domain annotation within the genus  A. baumannii compared to its largely a-pathogenic relatives in the Acinetobacter genus. Most A. baumannii isolates have T4aP with a pilus tip adhesin containing a protein domain variant not seen outside the pathogenic clade. This variant appears essential for bacterial motility and contributes to host cell adhesion and natural competence. However, some isolates have T4aP resembling those of largely a-pathogenic species in this genus. This indicates that the way these pili are used during infection processes differs between A. baumannii isolates probably as a consequence of niche adaptation. In a broader perspective, our findings highlight that many relevant genetic differences between pathogens and their a-pathogenic relatives emerge only on the domain- and sub-domain level. Thus, existing comparative genomics studies have likely uncovered only the tip of the iceberg of genetic determinants that contribute to A. baumannii virulence.Type IVa pili (T4aP) are hair-like, extendable, and retractable appendages that many bacteria use for interacting with their environment. Several human pathogens have independently recruited these pili for processes related to host infection, but the modifications necessary to turn T4aP into virulence factors are largely unknown. Here, we studied if and how T4aP components have changed in the nosocomial pathogen  Acinetobacter baumannii is a Gram negative nosocomial pathogen. A recent world-wide survey estimated that antimicrobial resistant A. baumannii strains were responsible for 10.4% of bacterial infections with fatal outcome in 2019  crystal violet.To analyze the piliation phenotype, cells were grown on LB agar plates at 37°C overnight. Cells were prepared for electron microscopy and visualized as previously described . Shadowi2 cell culture flasks using a humidified incubator with a 5% CO2 atmosphere at 37°C. HUVECs were seeded into six-well plates and infected with A. baumannii (MOI 50) for three hours. The supernatant was removed and cells with adherent bacteria were washed with PBS and detached using a cell scraper. Thereafter, adherent bacteria were quantified by plating serial dilution series. Visualisation and quantification of bacterial adhesion to human endothelial cells was done by fluorescence microscopy as described in . Authors need to reconsider the statement according to more recent evidence of the literature. I suggest the following three recent publications: Nowak, J. et al. (2017) J. Antimicrob. Chemother. 72, 3277–3282; Wong et al. 2017. Clin Microbiol Rev 30:409–447; Tacconelli et al. 2018. Lancet Infect Dis 18:318–327.2. Table 1. To avoid confusion, comP and pilA synonyms of pilin should be indicated both in the first column of the table as comP/ pilA instead then in thew footnote.3. Figure 1B and Figures 2 and 3. Inclusion of Acinetobacter oleivorans into Acinetobacter baumannii-calcoaceticus complex. Authors should indicate the methods/criteria used to include A. oleivorans into A. baumannii-calcoaceticus complex. While it is evident that A. olevoirans DR1 isolates is a distinct species of Acinetobacter genus :29-34), I did not find any A. oleivorans isolate identified at species level using MALDI-TOF  or Raman Spectrometry . Instead, it is possible to perform species identification of A. oleivorans genomes using ribosomal MLST . A collection of genomes assigned to 72 distinct species into Acinetobacter genus is available at in AcinetobacterPubMLST genomes databases athttps://pubmlst.org/bigsdb?db=pubmlst_abaumannii_isolates&page=query&genomes=1If identification at species level of Acinetobacter genomes included in the manuscript has been performed using genome sequences and/or ribosomalMLST allele profiles, I suggest to modify genomes classification by grouping A. baumannii, A. nosocomialis, A. pittii, A. seifertii and A. lactucae genomes into A. baumannii group and all other genomes into non-baumannii Acinetobacter group. This approach has been adopted using MALDI-TOF identification for Acinetobacter spp. .https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_isolates&page=query&genomes=14. Fig 3 International clonetypes of A. baumannii genomes. I have a major concern regarding assignment of A. baumannii genomes reported in Fig. 3. It has been shown by several publications that A. baumannii has a clonal population structure dominated by three global/international clonal lineages, named GC1, 2 and 3, and few additional epidemic clonal lineages, which emerged worldwide . Among several typing methods used, it has been demonstrated that the most appropriate to study A. baumannii genomes and assign to epidemic clonal lineages is PasteurMLST-based typing scheme . I invite Authors to assign A. baumannii genomes included in Figure 3 and in Supplementary Table 3 to international clonal types using Pasteur MLST scheme as described in Gaiarsa et al. Front. Microbiol. 10:930, 2019 and taking advantage of additional information found in AcinetobacterPubMLST genomes databases at Also, I invite Authors to correct international clonal type assignment of Figure 3. In fact, Acinetobacter baumannii TG215234 and Acinetobacter baumannii ATCC19606 are both assigned to international clonal type 2. This is true for Acinetobacter baumannii TG215234, while A. baumannii ATCC19606 has a distinct clonal type (assigned to PasteurST25 type). Moreover, I noticed that Authors considered in sdupplementary Table 3 an old version of A. baumannii ACICU genome sequence, while thay should consider the revised complete genome sequences of Acinetobacter baumannii strains AB307-0294 and ACICU belonging to global clones 1 and 2 published recently by Hamidian M et al. Insights from. Microb Genom. 2019 Oct;5(10):e000298.5. Domain architecture difference between FimT orthologs reported in Results section on lines 153-155. Authors should discuss whether this might be responsible for the absence of twitching motility in A. baumannii ACTCC 19606 strains observed in this study and in previous ones.6. Discussion, lines 350-362.Differences between ACICU and ATCC19606 PilA variants and comC – FimU genes should be explained by differences in genetic background of ACICU and ATCC 19606 genomes.7. Reference nr. 20. Please, correct title.Reviewer #2: This manuscript by Iruegas et al describes a pan-genomic comparison of type IV pilus (T4P) genes in the genus Acinetobacter. Based on this comparison, the authors found that for a specific cluster of those genes, recombination events had created substantial variation. In particular, the authors found the insertion of a VWA domain into comC, the tip adhesion, for some strains of A. baumannii, while others have the, apparently ancestral domain structure shared by A. baylyi. Alterations to this domain cause defects in multiple T4P functions in the A. baumannii AYE-T background.This manuscript is well-written and contains new knowledge for those with an interest in the function and evolutionary origins of T4P systems. Previous analysis of variation in Acinetobacter T4P systems has been focused on the major subunit, pilA, while the fimU/comC variation appears to be the result of a separate, possibly earlier, evolutionary divergence.The only significant concern I have with the interpretation of the data is how to interpret the loss of T4P function in the comCΔVWA and comCΔ166-256 mutants. While it is possible that the VWA structure plays a role in twitching motility, DNA-uptake and host-adherence, simpler explanation  is that ComC stabilizes the pilus structure and that the mutants are less stable than wild type ComC, leading to lower ComC expression and fewer pili.Specific comments:-Based on figures 1 and 3, it appears that pilV has similar levels of variation and may be covariant with comC and fimU, while only variation in the later two is discussed. Do the pilV genes in Var2, Var1-1 and Var1-2 subsets differ?-It is no fault of the authors because the naming of T4P genes and gene-products in the literature is a mess, but It may be easier for some readers to follow if the gene names of the Pseudomonas homologues  were used, simply because that system has been better studied historically. The authors do include both names in some parts of the text, but some genes  A simple table of equivalency could be added to Figure 1 to help readers more familiar with the Pseudomonas (rather than the A. baylyi) T4P nomenclature.-In Figure 1C, I am concerned that the figure overstates the differences between some of these genes. As the authors correctly point out, the absence of a pfam domain could simply be lack of recognition. I do not believe, for instance, there are any Acinetobacter fimT or pilX genes which lack the N-terminal PilD recognition site or TM helix which make up the N-Methyl domain. Similarly, I would imagine that all Acinetobacter fimU genes are gspH homologues based on the commonly proposed models for the T4Pa tip complex.-In Figure 4 (panels A and B), it is difficult to follow what is happening with the superimpositions at this figure resolution; however I am concerned that some of the differences are artifacts of attempting to align the whole multi-domain structure. Structure prediction (including AlphaFold) is much better at predicting domain structure than aligning multiple domains and for some  the domains may simply be rotated (arbitrarily) the wrong way in the model.Reviewer #3: This manuscript by Iruegas and colleagues deals with comparative genomics of T4aP systems in Acinetobacter genomes at the protein-domain level, in order to find determinants of pathogenicity in A. baumannii, whenever more classical comparative genomics involving gene repertoires could not find relevant traces. The authors claim to find some specificities in terms of domain compositions for pathogenic strains, and make some experimental testing on the role of these domains in abilities of Acinetobacter to adhere to human cells, to perform twitching motility or competence. The idea is appealing and interesting, as it is true that the information at the domain-level is very often overlooked in comparative genomics, even though it may hold crucial cues to understand bacterial adaptation. However, the manuscript is difficult to read and follow, as the bioinformatic methods in particular, and even the global approach is hard to grasp. In particular, it is unclear how protein domains were annotated (see my comments) and how a “domain loss” (or addition) was inferred, whenever it is crucial for the analysis that look into the evolution of the presence/absence pattern of protein domains. In the end, I am still wondering what really makes the difference between the sequences of the protein-domain variants of FimU and ComC. And can the outcome of a PFAM domain annotation suffice to infer the presence or absence of a functional domain? Considering the potential thresholds effects in such an analysis, I would assume that it should be completed with in-depth multiple sequence alignment analyses.It is also a bit difficult to understand how the recombination and protein domain analyses results articulate. I finally also wonder how reliable are the structural modelling experiments, and whether the experimental designs proposed to test the functional role of ComC domains highlighted by bioinformatic analyses, are sufficient to support the conclusions, in particular that the authors “provide experimental evidence that this finger conveys virulence-related functions in A. baumannii” .For these reasons, I unfortunately cannot be supportive of the publication of this article. I think the work and approach is valuable and of interest, but that there are missing pieces to make it a convincing case, and an effort to be made on how the results and methods are presented. A synthetic figure on the evolutionary proposed for example could be valuable. Please find more details in my comments below, I hope they will be considered as constructive.Major comments------------------1) Line 131. “Orthologs to all”... Please start the paragraph saying a word on the methodology for "ortholog search", otherwise the entire paragraph is hard to follow. As this is so central to this study’s methodology and results, I believe it should be explained what it consists in, and that providing a link to a Github repository is not sufficient in Methods (Line 396). Are you sure they are orthologs and follow the same gene history? How was it inferred? If not inferred, then maybe could the less precise "homologs" term be used?2) Line 152-153: rather than “chance effects” that sounds a bit odd, couldn’t the authors rather involve variations in sequences to explain the annotation or not of this domain with PFAM profiles? Depending on their specificity and sensitivity? On line 390 of the Methods, it would be very important to indicate what parameters were used for HMMER? Was the cu-ga option used? Otherwise, which thresholds? And which HMMER version?We touch here the heart of my main criticism: I do not understand how domains were annotated in details, and how/when a loss of domain was inferred, and how to distinguish it from cases where sequence divergence solely could explain the results.For example on lines 156-158 the authors then claim themselves “The domain loss in the A. baumannii type strain is caused by a substitution that results in a tyrosine at a position that is typically occupied by a leucine or an isoleucine in PF07963”. Rather than invoking here and elsewhere “domain losses”, could the authors rather involve threshold effects in the annotation? That the domain for example does not pass the threshold?Again, what is unclear to me here and later on, is whether the domain is really “lost” or gained  or whether the sequence is just divergent from the consensual domain as defined in PFAM. In Discussion (line 314-319), the authors themselves observe: “However, general features of a VWA domain are present also in ComC of a-pathogenic Acinetobacter isolates.” And further ask “Why then were these domains not annotated with the VWA_2 Pfam domain?”It probably does not change the functional interpretation, this domain diversification might be as well very functionally relevant, but it changes the angle of the overall approach here and the relevance of the underlying bioinformatic analyses.On lines 166, it is indicated that “We next investigated the evolutionary histories of ComC and FimU in greater detail.” However, I would have expected to see, aside from (or maybe even before) these phylogenetic analyses, an analysis of the multiple sequence alignment of these proteins first, because from the schemes on Fig 1C, it seems that there was not a clear-cut “domain” loss, as the sequences from both variants in both proteins show consistent length. Do they align well? If not, how could it impact the phylogeny? Do the variants clearly differ in sequence at the position of these domains? There is an alignment displayed in Fig4C, but it comes late in the article, and is limited to three sequences, making it difficult to infer how conserved are the different regions of the MSA.3) Fig. 2 represents a Maximum-Likelihood phylogeny, but neither branch lengths nor branch supports appear. Was there any attempt to root it? These reasons and questionings leave this tree difficult to use for evolutionary interpretations.4) A recombination analysis followed, but it is difficult to grasp how its results articulate with that of the domain analyses: recombination analyses showed a large tract of recombination spanning several genes  and yet in Fig S11, it is written in the legend “This indicates that a structural backbone of a VWA domain is modified, in a lineage-specific way, by insertions of different origins.” Could the authors clarify? How many events are invoked for instance?5) Regarding the recombination analysis, the paragraph should contain a word on which principle was used for recombination detection, to guide the authors through the results and drawn conclusions. Also, since the authors used phylogenetic approaches, could phylogenies of the genes inside/outside the cassette be used to demonstrate different evolutionary histories, hence supporting the cassette and recombination scenario? Could the phylogeny on Fig 3 already be it? I don’t see it much discussed in the main text. Also, the components from Fig S7 could have been used to complement Fig 3.6) Line 216-218: “Therefore, the two fingers are very likely of different evolutionary origins although their structural similarity and the shared presence of the Tyr-rich motif suggest that they originated from the same source” I found this sentence unclear and convoluted. Please rephrase.7) I am not a structural biologist, but what is the confidence of the 3D models obtained with AlphaFold in the precise regions of interest for ComC on Fig 4A? I have seen many times these models with mapped confidence scores. Are the features observed to differ between modelled “variants” reliable? What would happen were diverse sequences from a same “variant” chosen for the modelling? What is the confidence in the positioning of the finger-like protruding domain? Or its presence even? Unfortunately, additional information might be present on Fig S11, but the resolution of the image is too low. Could the “finger-like protrusion” region be somehow highlighted or pointed at where relevant on Fig 4?8) The proposed mutant comC to test experimentally are based on mutant proteins that seem somehow drastic versions of it. Despite the fact that 3D models predict similar folding, again what is the confidence for it?I am not convinced that the experimental results found in Fig. 5 demonstrate specifically the role of the VWA_2 PFAM (nor or the finger) domain in host cell adhesion, twitching motility or competence. They may demonstrate some role for the presence of the N-ter of the protein for the functioning of the T4aP, but I believe that by design the experiments cannot address the question on how the “content” of the N-ter may affect function. Some exchanges in N-ter experiments between variants, some point mutations of specific aa, or other experimental designs, may further contribute to address this question. For instance, what happens when performing the exact same experiments with corresponding fragments from the variant v2, or v1-2? Would they or not result in similar phenotypes? This should at least be discussed.Minor comments------------------9) Line 36, abstract section: “Both genes together form an evolutionary cassette” is not totally reflecting the scenario proposed by the authors, because the proposed cassette would contain several extra genes form the T4aP, right?10) I found the end of introduction too detailed on the results, it is somehow redundant with an abstract.11) I do not see the connection with the paragraph on lines 124-137 and the very 1st line of the paragraph "Acinetobacter T4aP components are best characterized in the naturally transformable bacterium A. baylyi ADP1". Should this sentence be placed elsewhere?12) Fig. 1B. it is unclear what should be compared here. Also, I do not get the meaning of the FAS_R score? Why do we need two scores here, and what is “the ortholog”? Please clarify to help the reader analyse the figure.13) Lines 149-150: “the architectures of four proteins, PilX, FimT, FimU, and ComC deviate to an extent between orthologs that it could indicate a change in function.” From Fig. 1B, one could also wonder about the relevance of ComO and ComN.14) Line 124: Included in what? Please rephrase as it is unclear.15) Line 170-171, it is mentioned that ComC and FimU are close in the genomes of Ab ATCC 19606 T is this also the case in other Acinetobacter genomes?16) Figure 2: what are the black branches?17) Line 185-186: “This indicates that recombination has affected the evolution of this locus” – I am not sure this can be argued at this point of the text. “might have affected” could be more appropriate?18) Line 386: "RefSeq database release 204" Could the authors mention on what date it was last accessed?Typos------Line 74 : remove «how »Line324: PDB means “Protein Data Bank”. Authors may want to add in text it is a database of structures of proteins?Line 413: where => were**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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This study clarified the effect of adding thermoresponsive xyloglucan on the bread-making properties and preservation of gluten-free rice-flour bread. The thickening polysaccharides used for preparing gluten-free rice-flour bread were modified tamarind gum , tamarind gum (TG), and xanthan gum (XT). The mechanical properties of the added polysaccharide thickener solutions and bread dough, the mechanical properties and sensory characteristics of rice-flour bread, and the aging properties of rice-flour bread were measured. The results showed that the MTG solution exhibited solification at 40 °C and gelation below 40 °C, which affected the dynamic viscoelasticity of the dough. The addition of MTG to gluten-free rice-flour bread reduced the specific volume, increased the moisture content, and reduced the stress at 70% compression. Therefore, the bread with MTG added was soft, moist, and preferred over other those with other additives. In terms of preservation, the addition of 0.5–0.75% of polysaccharides inhibited the hardening and aging of beard with MTG added. This indicates that the addition of MTG at low concentrations is effective in preserving gluten-free rice-flour breads. We found that the thickening polysaccharides had to be added in appropriate concentrations to improve the bread-making properties and achieve the preferred effect. The food self-sufficiency ratio in Japan has been declining, from 53% in 1985 to 37% in 2020 on a calorie basis  and fat Additionally, people suffering from food allergies in Japan account for 1–2% of the total population . Wheat iTo date, changing the viscosity of the batter by adding thickening agents such as polysaccharides ,15 or cePolysaccharide thickeners can impart viscosity, as well as cause gelatinization, enhance the emulsion stability, and enhance other food structure-forming features and the texture. Tamarind seed gum, a polysaccharide thickener, has been shown to enable thickening stabilization, gelation, and ice crystal stabilization and impart starch anti-retrogradation functions to various foods. Hence, tamarind seed gum has been studied for its bread-making properties ,26 and oThe rice flour used was leach rice flour , milled by wet-airflow milling . The particle size distribution of the rice flour was determined. The median, average, minimum, and maximum diameters of the rice-flour particles were 34.3, 45.5, 0.7, and 251.7 μm, respectively. The particle size distribution showed two peaks at approximately 10 and 50 μm. Granulated sugar , salt , olive oil , and dry yeast  were used as auxiliary ingredients. In addition, we used the thickening polysaccharides often used in processed foods: modified tamarind gum   were prepared by adding a small quantity of each polysaccharide in the powder form to pure distilled water; TG and XT were dissolved at 25 °C, and MTG was dissolved in distilled water at 5 °C or lower.The basic recipe for rice-flour bread batter required 8 g of granulated sugar, 1.5 g of salt, 1.5 g of dry yeast, 3 g of olive oil, and 90 g of distilled water per 100 g of rice flour. The polysaccharide thickener solutions were mixed with the rice flour at concentrations ranging from 0.25% to 1.0%. The amount of distilled water added ranged from 92.5 g (0.25% polysaccharide) to 100 g (1.0% polysaccharide), considering the water absorption rate of the polysaccharide thickening agent. For the rice-flour bread batter, 100 g of rice flour and each polysaccharide thickener solution (or 90 g distilled water for the control) were mixed using a mixer  at 35 °C and 150 rpm for 1 min. Subsequently, the batter was allowed to absorb water at 25 °C for 29 min. Granulated sugar, salt, and dry yeast were added, and the batter was kneaded at 150 rpm for 1 min. Olive oil was then added, following which the batter was kneaded at 150 rpm for 1 min. Finally, the batter was prepared by kneading for another 10 min at 150 rpm.The batter was first fermented for 30 min at 38 °C and 90% relative humidity (RH) in a whirlpool . It was then degassed and poured into a muffin mold  in 35 g portions using an electronic balance. This was followed by a second fermentation for 20 min under the same conditions mentioned. Subsequently, the rice-flour bread was baked in an oven  at 200 °C for 14 min. The bread was then cooled at 25 °C for 2 h, placed in a sealed polypropylene container, and stored at 4 °C and 65% RH for 0–3 days to be used for measurements.ϕ) and 7 mL of the sample were placed inside an aluminum cylinder (38 mmϕ) with holes, immersed in water, and left for 1 h to obtain a water absorption–time curve. The moisture content (g-water/g-dry matter) at the point when no change in water absorption was observed was considered the saturated moisture content. The water absorption was obtained from the difference between the initial and measured moisture contents of the rice flour.The water absorption and hydration rate of the rice flour were measured . A glassThe sample was prepared by combining the rice-flour sample with distilled water such that the sample concentration was 8% anhydrous equivalent and the total weight was 26 g. The stirring speed was 160 rpm. The pasting characteristics of rice flour were measured using the Rapid Visco-Analyzer . The analyzer used a program wherein the temperature was held at 50 °C for 1 min, increased to 95 °C over 7 min and maintained for 5 min, and then decreased to 50 °C over 6 min. A curve was acquired for a total of 19 min to obtain the pasting start temperature, peak viscosity and temperature, breakdown, setback, and final viscosity .The rice flour or polysaccharide thickener (powder) was fixed to a sample stand. Subsequently, Pt/Pd was deposited using an ion spatter  and observed using a tabletop scanning electron microscope  at an accelerating voltage of 15 kV and a magnification of 150–4000 times.The apparent viscosities of the polysaccharide thickener solution and prepared batter were measured using a cone-plate-type rotational viscometer . The value after 2 min was measured at 25 °C and rotational speeds of 3, 6, 12, 30, 60, and 100 rpm. The cone rotor measured 1°34′ × R24 and 3° × R12 for the polysaccharide thickener solution and bread batter, respectively.The dynamic viscoelasticities of the polysaccharide thickener solutions were measured using a Rheolograph Sol  with an amplitude of ±100 μm and a frequency of 3 Hz. The temperature dependence was measured using a 45 min program wherein the temperature was held at 5 °C for 5 min, increased to 80 °C over 15 min and maintained for 5 min, and then decreased to 5 °C over 15 min .The prepared rice-flour batter was measured using a dynamic viscoelasticity measuring device . A 25 mm diameter parallel plate fixture with a 1.0 mm gap was used. The strain dependence was measured at a frequency of 1.0 rad/s and 15 °C by varying the strain from 0.01% to 100% to identify the linear region. The temperature dependence was measured at a frequency of 1.0 rad/s and a strain rate of 0.1% between 15 and 50 °C. The dynamic viscoelasticities, storage modulus (G′), loss modulus (G″), and loss tangent (tanδ = G″/G′) were obtained .The bread was weighed after cooling at 25 °C. The apparent volume was measured by the rapeseed method , followiA sample (weighing approximately 3 g) from the crumb of the rice-flour bread was used to determine the moisture content using a halogen moisture meter . The measurement was based on the weight change over 120 s and was completed when the weight change was within 1 mg/s .The ratio of the moisture content on the day of measurement to that on the day of baking was determined to evaluate the moisture retention of bread.Cubes (20 mm × 20 mm × 20 mm) were cut from the crumb of the bread, and their rupture properties were measured using a creep meter . An acrylic resin disk plunger with a diameter of 40 mm was used to compress the sample to 99% of its height at a compression speed of 6 cm/min. Stresses at 25%, 40%, and 70% compression  were obtn = 3–7). The gluten-free rice-flour bread is semi-infinite solid, so the measurement conditions were as follows: light source, halogen lamp (D65); detector, photodiode; standard observer, 2°; measurement diameter, 10 mmϕ(Compliant with JIS Z8722).Cubes (20 mm × 20 mm × 20 mm) were cut from the crumb of the bread. The color was measured using the CIE L*a*b* color space ,39 and aSensory evaluation was conducted to assess the palatability of gluten-free rice-flour bread with a polysaccharide thickener. The samples were obtained after the bread was baked, cooled at 25 °C, placed in an airtight polypropylene container, and stored at 4 °C for 1 h.The sample was a muffin-shaped bread (7 cm in diameter at the top and 5 cm in diameter at the bottom) radially divided into 6 equal pieces, with each piece consisting of 3 slices. The 3 types of rice-flour bread with each polysaccharide thickening agent  were evaluated on a 7-point scale by a panel of 30 untrained female university students, with a control (no additive) as the standard. They were analytically evaluated based on the surface browning and gloss, texture, aroma when placed in the mouth, softness, stickiness, moistness, and residual feeling. Additionally, they were tested for preference based on the aroma when placed in the mouth, sweetness, aftertaste, and overall experience.The control and polysaccharide-thickened rice-flour breads were stored at 25 °C and 65% RH for 0–3 days. Subsequently, approximately 20 g of the crumb was ground with a mortar while dehydrating with approximately 60 mL ethanol and then filtered. This process was repeated twice. The sample was then defatted with acetone and air dried to obtain a defatted powder sample . The defStatistical processing of the measured objective characteristics of the samples and sensory evaluation results was conducted using the statistical analysis software Excel Statistics 2012. Further, one-way analysis of variance was performed, and the samples that exhibited significant differences were further tested by multiple comparisons using Scheffe’s method. The significance level for all tests was set to 5%.The purpose of this study was explained to the panel, and their understanding and consent were obtained before conducting the sensory evaluation. This study was carried out with the approval of the “Japan Women’s University Ethical Review Committee for Experimental Research on Human Subjects” .The water absorption characteristic curve of rice flour is shown in The RVA curve of rice flour is shown in The scanning electron micrographs of rice flour are shown in Particle sizes below 25 µm had a compound grain structure with small aggregates on the surface, whereas those larger than 25 µm had fewer small aggregates and a smooth surface.The flour particles exhibited a compound grain structure. It has been reported that rice flour milled by wet-airflow milling from the amyloplasts in cellular tissues has a starch grain structure . This stFurthermore, the scanning electron micrographs of the polysaccharide thickeners used in this study are shown in The storage modulus (G′) and loss tangent (tanδ) in the dynamic viscoelasticity of the polysaccharide thickener solutions are shown in The storage modulus of MTG increased when the temperature decreased from 80 to 40 °C but decreased upon cooling from approximately 40 to 5 °C. All concentrations of TG showed a constant storage modulus with increasing temperature and little change with decreasing temperature, while the storage modulus of XT increased with increasing concentration and decreasing temperature from 80 to 5 °C.The loss tangent, which represents the ratio of the loss modulus to the storage modulus, increased for MTG as the temperature decreased from 40 to 5 °C, with the viscous component particularly increasing at temperatures lower than 35 °C. The storage modulus increased as the temperature decreased from 75 to 40 °C, while the loss modulus increased from approximately 40 °C. The gelatinization of MTG at ~40 °C and its tendency to become a sol at temperatures below 40 °C were confirmed.It was also found that a concentration of more than 0.75% was required for the development of viscoelasticity in MTG. TG showed little change in both storage and loss modulus; hence, the change in behavior was also small for the loss tangent, but the viscous factor increased below 20 °C. XT showed that the loss tangent was almost constant irrespective of concentration and was shown to be less affected by temperature.−1.The temperature dependence of the dynamic viscoelasticity of the batter with each polysaccharide thickener is shown in These results indicate that the basic properties of the polysaccharide thickeners affect the characteristics of the batter: the TG system had a low viscosity close to that of the control when added to the batter, while the XT system had a high viscosity even at low concentrations. MTG was gelatinized at high temperatures and solubilized at low temperatures, suggesting that its sol–gel transition point is 35–40 °C. Reflecting this property, when MTG was added to the batter, the tanδ decreased at temperatures above 30 °C and the elastic factor increased, suggesting that the properties of the solution affected the batter.p < 0.05); the specific volume of bread with MTG and TG tended to be smaller than tha of bread with XT added. This is likely because the addition of polysaccharides increased the viscosity of the batter and prevented it from swelling.The specific volume and moisture content of gluten-free rice-flour bread baked with the polysaccharide thickeners are shown in p < 0.05), indicating that the water retention improved. This result suggests that the ability of the polysaccharide thickeners to prevent water release also had an effect on the bread by increasing the moisture content. The water-soluble polysaccharides used contain several hydroxyl groups, which improved water retention. TG has been reported to possess strong hydration and excellent water-retention properties [The moisture content was approximately 40% in the control. In contrast, the amount of water added increased as the quantity of polysaccharides added increased for the MTG-, TG-, and XT-added breads (operties . Comparep < 0.05). However, at 70% and 25% compression, the stress of the MTG-added bread decreased with increasing MTG concentration (p < 0.05), while the TG-added bread showed the lowest stress at 0.25% (p < 0.05).The stresses at 25%, 40%, and 70% compression for the gluten-free rice-flour bread baked with polysaccharide thickeners are shown in Changing the concentration was effective in improving the overall bread quality. However, the surface hardness of the bread was not affected by the addition of XT at any concentration, while the internal phase tended to be the hardest at 0.5% addition.p < 0.05), but there was no significant difference between the concentrations. The a* (redness) and b* (yellowness) values decreased with increasing polysaccharide concentration in the MTG-added bread (p < 0.05), but increased for the 0.25% TG-added bread and decreased at higher concentrations of TG (p < 0.05). The b* values of the XT-added bread decreased at 0.25% and 0.5% additions (p < 0.05), showing a different trend from that of the TG-added bread.The chromaticity on the surface of the gluten-free rice-flour bread baked with the polysaccharide thickeners is shown in The color of the bread showed a tendency for redness and yellowness to decrease with increasing concentrations of TG and MTG additions, which differed for XT. Regarding the color of gluten-free rice-flour bread, it has been reported that the L and a values are lower when the specific volume of the bread is lower . This fap < 0.05). The results are shown in p < 0.05), and the XT-added bread was evaluated to be finer in grain than the control (p < 0.05). Furthermore, the additives were expected to mask the flavor of rice flour. The flavor of the TG-added bread was significantly weaker than that of the control (p < 0.05), but the flavor of the MTG- and XT-added bread was similar to that of the control. The XT-added bread was the softest, followed by the MTG-added bread (p < 0.05). No significant difference in softness were found between the TG-added bread and the control. The moistness of the bread was evaluated to be greater than that of the control for all polysaccharide thickeners (p < 0.05). Additionally, the MTG-added bread was preferred over the control in terms of sweetness (p < 0.05), aftertaste (p < 0.05), and overall preference evaluation (p < 0.05).Sensory evaluation was conducted using the grading method to evaluate the palatability of rice-flour bread with added polysaccharides. Samples with 0.5% addition of either the MTG, TG, or XT polysaccharide thickener and bread without additives were evaluated. Higher scores of the samples were considered to render them superior to the control for all parameters except the baking color and flavor (The effect of storage on the moisture content of gluten-free rice-flour bread with added polysaccharides is shown in p < 0.05). The MTG-added bread was softer than the control until day 2 at 0.5% MTG concentration and until day 1 at 0.75% MTG concentration, indicating that the addition of MTG inhibited the hardening of the inner phase of the bread. The TG-added bread showed the lowest value at 0.5% TG concentration, which suppressed the hardening of the bread. Furthermore, the XT-added bread showed a smaller value at 1.0% XT concentration than did the control and TG-added bread, which suppressed the hardening of the bread. The values of the XT-added bread indicated that higher concentrations have a greater effect on hardening inhibition throughout the 3-day storage period.To investigate whether retrogradation caused the hardening of the bread, the relative intensities of gluten-free rice-flour bread with 0.5% polysaccharide thickener were calculated from the X-ray diffraction pattern  and the finer the grain texture (p < 0.1)  .p < 0.01). A higher moisture content indicated that the bread was softer and moister (p < 0.1)  .These results indicate that the type and concentration of polysaccharide thickeners added to gluten-free rice-flour bread have different effects on their bread-making and preservation properties. In terms of shelf life, the addition of 0.5–0.75% of polysaccharides inhibited the hardening and retrogradation of MTG-added bread. In comparison, the addition of TG to bread at a lower concentration of 0.5% had an inhibitory effect on hardening at 25% compressive strength, while XT-added breads required a 1.0% concentration.The addition of MTG improved the bread palatability and the bread-making and shelf-life properties of gluten-free rice-flour bread. In addition, a positive correlation was observed between the viscosity coefficient of the batter and the texture and softness of the bread, suggesting that the viscosity coefficient of the batter is important for controlling the texture and softness of gluten-free rice-flour bread. Sensory evaluations revealed that adding polysaccharide thickeners improved the overall consumption experience owing to their various effects, such as increasing the moisture content of the breads. It was suggested that the viscosity coefficient of the batter can be used to control the grain texture and softness of gluten-free rice-flour bread, and the quality of the final product could be regulated by controlling the composition of the batter.Gluten-free rice-flour bread hardens over time and possesses poor storage qualities, which are attributed to its short shelf life of only 3 days. In this study, the type and concentration of the added polysaccharide thickener were found to exert different effects on the bread-making properties and shelf life. Results indicated that the addition of 0.5% MTG/TG or 1% XT effectively prevented hardening. Further, the addition of TG or MTG to gluten-free rice-flour bread was more effective in bread making and preservation at lower concentrations than the addition of XT. MTG addition reduced the specific volume of the bread but increased the moisture content and retention. It also reduced the stress at 70% compression and was found to be soft, moist, and preferred over the other additives and control, demonstrating the positive impact of the thermoresponsive xyloglucan additive. Further investigations into the bread-making properties of gluten-free rice-flour bread are essential for improving preferable qualities and preservation characteristics, as they could greatly help people suffering from wheat-flour and other allergies, as well as celiac disease, by providing good alternative breads and baked goods based on rice flour.Further, we believe that this paper will be of interest to the readership of this journal because people suffer from wheat-flour and other allergies.