Journal of Molecular Biology最新文献

All tRNAs undergo a series of chemical modifications to fold and function correctly. In mammals, the C32 nucleotide in the anticodon loop of tRNA-Arg-CCU and UCU is methylated to form 3-methylcytosine (m3C). Deficiency of m3C in arginine tRNAs has been linked to human neurodevelopmental disorders, indicating a critical biological role for m3C modification. However, the structural repercussions of m3C modification are not well understood. Here, we examine the structural effects of m3C32 modification on the anticodon stem loop (ASL) of human tRNA-Arg-UCU-4-1, a unique tRNA with enriched expression in the central nervous system. Optical melting experiments demonstrate that m3C modification can locally disrupt nearby base pairing within the ASL while simultaneously stabilizing the ASL electrostatically, resulting in little net change thermodynamically. The isoenergetic nature of the C32-A38 pair versus the m3C32-A38 pair may help discriminate against structures not adopting canonical C32-A38 pairings, as most other m3C pairings are unfavorable. Furthermore, multidimensional NMR reveals that after m3C modification there are changes in hairpin loop structure and dynamics, the structure of A37, and the neighboring A31-U39 base pair. However, these structural changes after modification are made while maintaining the shape of the C32-A38 pairing, which is essential for efficient tRNA function in translation. These findings suggest that m3C32 modification could alter interactions of tRNA-Arg isodecoders with one or more binding partners while simultaneously maintaining the tRNA's ability to function in translation.

The structure and function of transfer RNAs (tRNAs) are highly dependent on post-transcriptional chemical modifications that attach distinct chemical groups to various nucleobase atoms at selected tRNA positions via enzymatic reactions. In all three domains of life, the greatest diversity of chemical modifications is concentrated at positions 34 and 37 of the tRNA anticodon loops. N⁶-threonylcarbamoyladenosine (t⁶A) is an essential and universal modification occurring at position 37 of tRNAs that decode codons beginning with an adenine. In a subset of tRNAs from specific organisms, t⁶A is converted into a variety of hypermodified forms, including cyclic N⁶-threonylcarbamoyladenosine (ct⁶A), hydroxy-N⁶-threonylcarbamoyladenosine (ht⁶A), N⁶-methyl-N⁶-threonylcarbamoyladenosine (m⁶t⁶A), 2-methylthio-N⁶-threonylcarbamoyladenosine (ms²t⁶A) and 2-methylthio-cyclic N⁶-threonylcarbamoyladenosine (ms²ct⁶A). The tRNAs carrying t⁶A or one of its hypermodified derivatives are dubbed as the t⁶A family. The t⁶A family modifications pre-organize the anticodon loop in a conformation that enhances binding to the cognate mRNA codons, thereby promoting translational fidelity. The dysfunctional installation of modifications in the tRNA t⁶A family leads to translation errors, compromises proteostasis and cell viability, interferes with the growth and development of higher eukaryotes and is implicated in several human diseases, such as neurological disorders, mitochondrial encephalomyopathies, type 2 diabetes and cancers. In addition, loss-of-function mutations in KEOPS complex-the tRNA t⁶A-modifying enzyme-are associated with shortened telomeres, defects in DNA damage response and transcriptional dysregulation in eukaryotes. The chemical structures, the molecular functions, the known cellular roles and the biosynthetic pathways of the t⁶A tRNA family are described by integrating and linking biochemical and structural data on these modifications to their biological functions.

Kallikrein-related peptidases (KLKs) have garnered significant interest in the field of biomedical research due to their diverse roles in various physiological and pathological processes. However, the structurally conserved active site of the KLK family presents challenges for the development of specific inhibitors. Given the pro-tumorigenic roles KLKs play in various cancers, identifying new avenues for specific inhibition is therefore vital. Here, we introduce a novel approach to target a distinct KLK4 sequence by a unique immunization approach for monoclonal antibody generation, targeting loop 3, a region of high sequence and structural diversity as a candidate for allosteric control of KLK4 activity. Immunisation produced an antibody capable of interacting with both KLK4 and loop 3 with high affinity, which inhibited KLK4 proteolytic activity, and hindered proliferation and migration in ovarian cancer cell lines. Encouragingly, its potential application extends to preclinical models characterized by KLK4 overexpression. Our findings underscore the promise of this novel approach to addressing the challenges of specifically inhibiting ubiquitous serine proteases, with particular relevance to targeting KLK4, a protease instrumental in the progression of ovarian carcinoma and other cancer types.

Most of the knowledge on the mechanisms and functions of autophagy originates from studies in yeast and other cellular models. How this valuable information is translated to the brain, one of the most complex and evolving organs, has been intensely investigated. Fueled by the tight dependence of the mammalian brain on autophagy, and the strong links of human brain diseases with autophagy impairment, the field has revealed adaptations of the autophagic machinery to the physiology of neurons and glia, the highly specialized cell types of the brain. Here, we first provide a detailed account of the tools available for studying brain autophagy; we then focus on the recent advancements in understanding how autophagy is regulated in brain cells, and how it contributes to their homeostasis and integrated functions. Finally, we discuss novel insights and open questions that the new knowledge has raised in the field.

N¹-methyladenosine (m¹A) is a conserved modification on house-keeping RNAs, including tRNAs and rRNAs. With recent advancement on m¹A detection and mapping, m¹A is revealed to have a secret life with regulatory functions. This includes the regulation of its canonical substrate tRNAs, and expands into new territories such as tRNA fragments, mRNAs and repeat RNAs. The dynamic regulation of m¹A has been shown in different biological contexts, including stress response, diet, T cell activation and aging. Interestingly, m¹A can also be installed by non-enzymatic mechanisms. However, technical challenges remain in m¹A site mapping; as a result, controversies have been observed across different labs or different methods. In this review we will summarize the recent development of m¹A detection, its dynamic regulation, and its biological functions on diverse RNA substrates.