Pharmacological profiling of a berbamine derivative for lymphoma treatment.

ABSTRACT: Ca2+/calmodulin-dependent protein kinase II γ (CAMKIIγ) has been identified as a potential target for treating cancer. Based on our previous study of berbamine (BBM) as a CAMKIIγ inhibitor, we have synthesized a new BBM derivative termed PA4. Compared with BBM, PA4 showed improved potency and specificity and was more cytotoxic against lymphoma and leukemia than against other types of cancer. In addition to indirectly targeting c-Myc protein stability, we demonstrated that its cytotoxic effects were also mediated via increased reactive oxygen species production in lymphoma cells. PA4 significantly impeded tumor growth in vivo in a xenograft T-cell lymphoma mouse model. Pharmacokinetics studies demonstrated quick absorption into plasma after oral administration, with a maximum concentration of 1680 ± 479 ng/mL at 5.33 ± 2.31 hours. The calculated oral absolute bioavailability was 34.1%. Toxicity assessment of PA4 showed that the therapeutic window used in our experiments was safe for future development. Given its efficacy, safety, and favorable pharmacokinetic profile, PA4 is a potential lead candidate for treating lymphoma.

Anti-adipogenic properties of clock activator chlorhexidine and a new derivative.

Background: The circadian clock exerts temporal control of metabolic pathways to maintain homeostasis, and its disruption leads to the development of obesity and insulin resistance. In adipose tissue, key regulators of clock machinery orchestrate adipogenic processes via the Wnt signaling pathway to impact mature adipocyte development. Methods: Based on the recent finding of chlorhexidine as a new clock activator, we determined its potential anti-adipogenic activities in distinct adipogenic progenitor models. Furthermore, we report the structural optimization of chlorhexidine leading to the discovery of analogs with improved efficacy in inhibiting adipogenesis. Results: In adipogenic progenitors with Per2::dLuc luciferase reporter, Chlorhexidine shortened clock period length with induction of core clock components. Consistent with its clock-activating function, Chlorhexidine robustly suppressed the lineage commitment and maturation of adipogenic mesenchymal precursors, with comparable effect on inhibiting preadipocyte terminal differentiation. Mechanistically, we show that Chlorhexidine induces signaling components of the Wnt pathway resulting in activation of Wnt activity. Via modification of its chemical scaffold, we generated analogs of chlorhexidine that led to the identification of CM002 as a new clock- activating molecule with improved anti-adipogenic activity. Conclusions: Collectively, our findings uncovered the anti-adipogenic functions of a new class of small molecule clock activators. These compounds provide novel chemical probes to dissect clock function in maintaining metabolic homeostasis and may have therapeutic implications in obesity and associated metabolic disorders.

CYP8B1 downregulation mediates the metabolic effects of vertical sleeve gastrectomy in mice.

BACKGROUND AND AIMS: Although the benefits of vertical sleeve gastrectomy (VSG) surgery are well known, the molecular mechanisms by which VSG alleviates obesity and its complications remain unclear. We aim to determine the role of CYP8B1 (cytochrome P450, family 8, subfamily B, polypeptide 1) in mediating the metabolic benefits of VSG. APPROACH AND RESULTS: We found that expression of CYP8B1, a key enzyme in controlling the 12α-hydroxylated (12α-OH) bile acid (BA) to non-12α-OH BA ratio, was strongly downregulated after VSG. Using genetic mouse models of CYP8B1 overexpression, knockdown, and knockout, we demonstrated that overexpression of CYP8B1 dampened the metabolic improvements associated with VSG. In contrast, short hairpin RNA-mediated CYP8B1 knockdown improved metabolism similar to those observed after VSG. Cyp8b1 deficiency diminished the metabolic effects of VSG. Further, VSG-induced alterations to the 12α-OH/non-12α-OH BA ratio in the BA pool depended on CYP8B1 expression level. Consequently, intestinal lipid absorption was restricted, and the gut microbiota (GM) profile was altered. Fecal microbiota transplantation from wild type-VSG mice (vs. fecal microbiota transplantation from wild-type-sham mice) improved metabolism in recipient mice, while there were no differences between mice that received fecal microbiota transplantation from knockout-sham and knockout-VSG mice. CONCLUSIONS: CYP8B1 is a critical downstream target of VSG. Modulation of BA composition and gut microbiota profile by targeting CYP8B1 may provide novel insight into the development of therapies that noninvasively mimic bariatric surgery to treat obesity and its complications.

Four Kinds of Polymer Microspheres Prepared by the Seed Swelling Method Used to Purify the Industrial Production of Phytol.

Four monodisperse porous polymer microspheres were successfully prepared by seed emulsion polymerization and used as stationary phases for HPLC and preparative high-performance liquid chromatography (Prep-HPLC). All four polymer microspheres(polystyrene-polystyrene (PS-PS), polystyrene-poly(glycidyl methylate) (PS-PGMA), polystyrene-poly(methyl methylate) and poly(glycidyl methylate)-poly(glycidyl methylate) were used for filling HPLC empty columns. According to the analysis results of the HPLC column, PS-PS and PS-PGMA microspheres were screened out as the stationary phase of Prep-HPLC. The industrial-grade phytol was successfully separated and purified, and the purity of the final phytol was as high as 99%. The two types of polymer microspheres have been applied to industrial-grade phytol purification and have been used in factories.

Downregulation of hepatic lncRNA Gm19619 improves gluconeogenesis and lipogenesis following vertical sleeve gastrectomy in mice.

Long non-coding RNAs (lncRNAs) are emerging important epigenetic regulators in metabolic processes. Whether they contribute to the metabolic effects of vertical sleeve gastrectomy (VSG), one of the most effective treatments for sustainable weight loss and metabolic improvement, is unknown. Herein, we identify a hepatic lncRNA Gm19619, which is strongly repressed by VSG but highly up-regulated by diet-induced obesity and overnight-fasting in mice. Forced transcription of Gm19619 in the mouse liver significantly promotes hepatic gluconeogenesis with the elevated expression of G6pc and Pck1. In contrast, AAV-CasRx mediated knockdown of Gm19619 in high-fat diet-fed mice significantly improves hepatic glucose and lipid metabolism. Mechanistically, Gm19619 is enriched along genomic regions encoding leptin receptor (Lepr) and transcription factor Foxo1, as revealed in chromatin isolation by RNA purification (ChIRP) assay and is confirmed to modulate their transcription in the mouse liver. In conclusion, Gm19619 may enhance gluconeogenesis and lipid accumulation in the liver.

Analysis of appendicitis management during COVID-19 pandemic: A study of Chinese adult cohorts.

Background: Healthcare seeking behavior has been widely impacted due to the restricted movements of individuals during the Coronavirus disease-19 (COVID-19) pandemic. This study aims to perform risk stratification in patients requiring timely intervention during the recovery periods. Methods: Operation notes of acute appendicitis (AA) patients within a hospital were analyzed during three six-month periods (23 January-23 July in 2019, 2020, and 2021, respectively). Patient data were collected retrospectively including demographics, pre-emergency status, perioperative information, postoperative outcomes, and follow-up results. Results: 321 patients were included in this study, with 111, 86, and 124 patients in 2019, 2020, and 2021 groups, respectively. The median age of patients decreased by 4 years in 2020 as compared to that in 2019. The proportion of pre-hospitalization symptoms duration of more than 48 h in the 2020 group was higher (36.05% in 2020 vs. 22.52% in 2019). Length of hospital stay (LOS) in 2020 was shorter than it was during the same period in 2019 (4.77 vs. 5.64) and LOS in 2021 was shorter than in 2019 (4.13 vs. 5.64). Compared to the lockdown period, the proportion of patients with recurrent AA was higher in the post-lockdown period (15.1% vs. 27.4%). The median age was 34 years (vaccinated) vs. 37 years (unvaccinated). Logistic regression suggests that elevated C-reactive protein (CRP) (OR = 1.018, CI = 1.010-1.028), white cell count (WBC) (OR = 1.207, CI = 1.079-1.350), female (OR = 2.958, CI = 1.286-6.802), recurrent (OR = 3.865, CI = 1.149-12.997), and fecalith (OR = 2.308, CI = 1.007-5.289) were associated with complicated appendicitis (CA). Conclusion: The lockdown measures during the COVID-19 epidemic are shown to be correlated with a reduction in the proportion of AA patients who underwent surgery, particularly in older adults. Risk factors for CA include elevated CRP, WBC, female, recurrent, and fecalith.

Inhibition of the CDK2 and Cyclin A complex leads to autophagic degradation of CDK2 in cancer cells.

Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.

Intestinal AMPK modulation of microbiota mediates crosstalk with brown fat to control thermogenesis.

The energy-dissipating capacity of brown adipose tissue through thermogenesis can be targeted to improve energy balance. Mammalian 5'-AMP-activated protein kinase, a key nutrient sensor for maintaining cellular energy status, is a known therapeutic target in Type II diabetes. Despite its well-established roles in regulating glucose metabolism in various tissues, the functions of AMPK in the intestine remain largely unexplored. Here we show that AMPKα1 deficiency in the intestine results in weight gain and impaired glucose tolerance under high fat diet feeding, while metformin administration fails to ameliorate these metabolic disorders in intestinal AMPKα1 knockout mice. Further, AMPKα1 in the intestine communicates with brown adipose tissue to promote thermogenesis. Mechanistically, we uncover a link between intestinal AMPKα1 activation and BAT thermogenic regulation through modulating anti-microbial peptide-controlled gut microbiota and the metabolites. Our findings identify AMPKα1-mediated mechanisms of intestine-BAT communication that may partially underlie the therapeutic effects of metformin.

Bile Acid-Mediated Activation of Brown Fat Protects From Alcohol-Induced Steatosis and Liver Injury in Mice.

BACKGROUND & AIMS: Alcohol-associated liver disease (AALD) is one of the most common causes of liver injury and failure. Limited knowledge of the mechanisms underlying AALD impedes the development of efficacious therapies. Bile acid (BA) signaling was shown to participate in the progression of AALD. However, the mechanisms remain poorly understood. METHODS: C57BL/6J wild-type (WT), Takeda G-protein-coupled bile acid receptor 5 (TGR5) knockout (KO) and brown adipose tissue (BAT)-specific TGR5 knockdown mice were subjected to ethanol feeding-induced AALD. Liver samples from alcoholic hepatitis patients were used to examine the BA circulation signaling. Human Embryonic Kidney Cells 293 were used for the TGR5 reporter assay. 23(S)-methyl-lithocholic acid was used as a molecular tool to confirm the regulatory functions of BAT in the AALD mouse model. RESULTS: Ethanol feeding increased the expression of the thermogenesis genes downstream of TGR5 in BAT of WT, but not TGR5 KO, mice. TGR5 deficiency significantly blocked BAT activity and energy expenditure in mice after ethanol feeding. Alcohol increased serum BA levels in mice and human beings through altering BA transportation, and the altered BAs activated TGR5 signaling to regulate metabolism. Compared with ethanol-fed WT mice, ethanol-fed TGR5 KO mice showed less free fatty acid (FFA) ß-oxidation in BAT, leading to higher levels of FFA in the circulation, increased liver uptake of FFAs, and exacerbated AALD. BAT-specific TGR5 knockdown mice showed similar results with TGR5 KO mice in AALD. Agonist treatment significantly activated TGR5 signaling in BAT, increased thermogenesis, reduced serum FFA level, and ameliorated hepatic steatosis and injury in AALD mice, while these effects were lost in TGR5 KO mice. CONCLUSIONS: BA signaling plays a protective role in AALD by enhancing BAT thermogenesis. Targeting TGR5 in BAT may be a promising approach for the treatment of AALD.

Vertical sleeve gastrectomy confers metabolic improvements by reducing intestinal bile acids and lipid absorption in mice.

Vertical sleeve gastrectomy (VSG) is one of the most effective and durable therapies for morbid obesity and its related complications. Although bile acids (BAs) have been implicated as downstream mediators of VSG, the specific mechanisms through which BA changes contribute to the metabolic effects of VSG remain poorly understood. Here, we confirm that high fat diet-fed global farnesoid X receptor (Fxr) knockout mice are resistant to the beneficial metabolic effects of VSG. However, the beneficial effects of VSG were retained in high fat diet-fed intestine- or liver-specific Fxr knockouts, and VSG did not result in Fxr activation in the liver or intestine of control mice. Instead, VSG decreased expression of positive hepatic Fxr target genes, including the bile salt export pump (Bsep) that delivers BAs to the biliary pathway. This reduced small intestine BA levels in mice, leading to lower intestinal fat absorption. These findings were verified in sterol 27-hydroxylase (Cyp27a1) knockout mice, which exhibited low intestinal BAs and fat absorption and did not show metabolic improvements following VSG. In addition, restoring small intestinal BA levels by dietary supplementation with taurocholic acid (TCA) partially blocked the beneficial effects of VSG. Altogether, these findings suggest that reductions in intestinal BAs and lipid absorption contribute to the metabolic benefits of VSG.

Comprehensive analysis of the expression and prognostic value of CXC chemokines in colorectal cancer.

The C-X-C motif (CXC) chemokines play an important role in inflammatory processes and angiogenesis and are also associated with tumor development, progression and metastasis. They can be either promoting or inhibiting factors in colorectal cancers (CRC). The expression patterns and prognostic values of the CXC family still need further investigation. In this study, we investigated data related to transcription, translation, survival and tumor immune infiltration for CXC chemokines in patients with CRC from the ONCOMINE, GEPIA, cBioPortal, HPA and TIMER databases. We found that the expression levels of CXCL1-3, CXCL5, and CXCL8 were higher in CRC tissues than in colorectal tissues. Expression among stages significantly varied for CXCL1-3 and CXCL9-11. The survival analysis revealed that high transcriptional levels of CXCL4 and CXCL9-11 could serve as positive prognostic factors for patients with CRC. CXCL9-11 were highly associated with CD8+ T cells and natural killer (NK) cells in the tumor immune infiltration analysis, indicating their role in the antitumor immune response. This study implies that CXCL1-3, CXCL5, and CXCL8 are important factors during CRC oncogenesis and that CXCL9-11 could be new biomarkers for the prognosis of CRC.

Tumor-suppressive function and mechanism of HOXB13 in right-sided colon cancer.

Right-sided colon cancer (RCC) and left-sided colon cancer (LCC) differ in their clinical and molecular features. An investigation of differentially expressed genes (DEGs) between RCC and LCC could contribute to targeted therapy for colon cancer, especially RCC, which has a poor prognosis. Here, we identified HOXB13, which was significantly less expressed in RCC than in LCC and associated with prognosis in RCC, by using 5 datasets from the Gene Expression Omnibus (GEO). Tissue sample analysis showed that HOXB13 was differentially expressed between normal and only RCC tumor tissues. HOXB13 inhibited colon cancer cell proliferation and induced apoptosis both in vitro and in vivo. Furthermore, we found that HOXB13 might be regulated by DNMT3B and suppress C-myc expression to exert antitumor effects via ß-catenin/TCF4 signals in RCC. In conclusion, the current study is the first to demonstrate that HOXB13 has a tumor-suppressive effect in RCC. High expression levels of HOXB13 are associated with prolonged overall survival in patients with RCC. The DNMT3B-HOXB13-C-myc signaling axis might be a molecular target for the treatment of RCC.

Bile-ology: from bench to bedside.

Bile acids (BAs) are originally known as detergents essential for the digestion and absorption of lipids. In recent years, extensive research has unveiled new functions of BAs as gut hormones that modulate physiological and pathological processes, including glucose and lipid metabolism, energy expenditure, inflammation, tumorigenesis, cardiovascular disease, and even the central nervous system in addition to cholesterol homeostasis, enterohepatic protection and liver regeneration. BAs are closely linked with gut microbiota which might explain some of their crucial roles in organs. The signaling actions of BAs can also be mediated through specific nuclear receptors and membrane-bound G protein-coupled receptors. Several pharmacological agents or bariatric surgeries have demonstrated efficacious therapeutic effects on metabolic diseases through targeting BA signaling. In this mini-review, we summarize recent advances in bile-ology, focusing on its translational studies.

Targeting Bile Acid-Activated Receptors in Bariatric Surgery.

Bariatric surgical procedures, including Roux-en-Y gastric bypass and vertical sleeve gastrectomy, are currently the most effective clinical approaches to achieve a significant and sustainable weight loss. Bariatric surgery also concomitantly improves type 2 diabetes and other metabolic diseases such as nonalcoholic steatohepatitis, cardiovascular diseases, and hyperlipidemia. However, despite the recent exciting progress in the understanding how bariatric surgery works, the underlying molecular mechanisms of bariatric surgery remain largely unknown. Interestingly, bile acids are emerging as potential signaling molecules to mediate the beneficial effects of bariatric surgery. In this review, we summarize the recent findings on bile acids and their activated receptors in mediating the beneficial metabolic effects of bariatric surgery. We also discuss the potential to target bile acid-activated receptors in order to treat obesity and other metabolic diseases.

Self-protective responses to norvaline-induced stress in a leucyl-tRNA synthetase editing-deficient yeast strain.

The editing function of aminoacyl-tRNA synthetases (aaRSs) is indispensible for formation of the correct aminoacyl-tRNAs. Editing deficiency may lead to growth inhibition and the pathogenesis of various diseases. Herein, we confirmed that norvaline (Nva) but not isoleucine or valine is the major threat to the editing function of Saccharomyces cerevisiae leucyl-tRNA synthetase (ScLeuRS), both in vitro and in vivo. Nva could be misincorporated into the proteome of the LeuRS editing-deficient yeast strain (D419A/ScΔleuS), potentially resulting in dysfunctional protein folding and growth delay. Furthermore, the exploration of the Nva-induced intracellular stress response mechanism in D419A/ScΔleuS revealed that Hsp70 chaperones were markedly upregulated in response to the potential protein misfolding. Additionally, proline (Pro), glutamate (Glu) and glutamine (Gln), which may accumulate due to the conversion of Nva, collectively contributed to the reduction of reactive oxygen species (ROS) levels in Nva-treated D419A/ScΔleuS cells. In conclusion, our study highlights the significance of the editing function of LeuRS and provides clues for understanding the intracellular stress protective mechanisms that are triggered in aaRS editing-deficient organisms.

Translational Quality Control by Bacterial Threonyl-tRNA Synthetases.

Translational fidelity mediated by aminoacyl-tRNA synthetases ensures the generation of the correct aminoacyl-tRNAs, which is critical for most species. Threonyl-tRNA synthetase (ThrRS) contains multiple domains, including an N2 editing domain. Of the ThrRS domains, N1 is the last to be assigned a function. Here, we found that ThrRSs from Mycoplasma species exhibit differences in their domain composition and editing active sites compared with the canonical ThrRSs. The Mycoplasma mobile ThrRS, the first example of a ThrRS naturally lacking the N1 domain, displays efficient post-transfer editing activity. In contrast, the Mycoplasma capricolum ThrRS, which harbors an N1 domain and a degenerate N2 domain, is editing-defective. Only editing-capable ThrRSs were able to support the growth of a yeast thrS deletion strain (ScΔthrS), thus suggesting that ScΔthrS is an excellent tool for studying the in vivo editing of introduced bacterial ThrRSs. On the basis of the presence or absence of an N1 domain, we further revealed the crucial importance of the only absolutely conserved residue within the N1 domain in regulating editing by mediating an N1-N2 domain interaction in Escherichia coli ThrRS. Our results reveal the translational quality control of various ThrRSs and the role of the N1 domain in translational fidelity.

A Newly Identified Missense Mutation in FARS2 Causes Autosomal-Recessive Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by spasticity of the lower limbs due to pyramidal tract dysfunction. Here, we report that a missense homozygous mutation c.424G>T (p.D142Y) in the FARS2 gene, which encodes a mitochondrial phenylalanyl tRNA synthetase (mtPheRS), causes HSP in a Chinese consanguineous family by using combination of homozygous mapping and whole-exome sequencing. Immunohistochemical experiments were performed showing that the FARS2 protein was highly expressed in the Purkinje cells of rat cerebellum. The aminoacylation activity of mtPheRS was severely disrupted by the p.D142Y substitution in vitro not only in the first aminoacylation step but also in the last transfer step. Taken together, our results indicate that a missense mutation in FARS2 contributes to HSP, which has the clinical significance of the regulation of tRNA synthetases in human neurodegenerative diseases.

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu.

Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNA(Leu) formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNA(Leu) through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNA(Ser) (CatRNA(Ser)(CAG)) in addition to its cognate CatRNA(Leu), leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNA(Ser), suggesting the significance of their highly divergent CTDs in tRNA(Ser) recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNA(Ser) and cognate CatRNA(Leu). Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain.

Degenerate connective polypeptide 1 (CP1) domain from human mitochondrial leucyl-tRNA synthetase.

The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with noncognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain that has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different noncognate amino acids are not completely understood. Here, we identified the essential role of the evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, a broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for noncognate norvaline (Nva) and valine (Val). hmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Moreover, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity and acquired fidelity during aminoacylation. This work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency.

Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2'-O-methyltransferase.

Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNA(Leu)CAA and tRNA(Leu)UAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2'-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i(6)) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2'-O-methylation relies on prior N(6)-isopentenyladenosine modification at position 37.