The clinical success of immunotherapy has revolutionized the treatment of cancer patients, bringing renewed attention to tumor-infiltrating lymphocytes (TILs) of various cancer types. Immune checkpoint blockade is effective in patients with mismatched repair defects and high microsatellite instability (dMMR-MSI-H) in metastatic colorectal cancer (CRC), leading the FDA to accelerate the approval of two programmed cell death 1 (PD-1) blocking antibodies, pembrolizumab and nivolumab, for treatment of dMMR-MSI-H cancers. In contrast, patients with proficient mismatch repair and low levels of microsatellite stability or microsatellite instability (pMMR-MSI-L/MSS) typically have low tumor-infiltrating lymphocytes and have shown unsatisfied responses to the immune checkpoint inhibitor. Different TILs environments reflect different responses to immunotherapy, highlighting the complexity of the underlying tumor-immune interaction. Profiling of TILs fundamental Indication would shed light on the mechanisms of cancer-immune evasion, thus providing opportunities for the development of novel therapeutic strategies. In this review, we summarize phenotypic diversities of TILs and their connections with prognosis in CRC and provide insights into the subsets-specific nature of TILs with different MSI status. We also discuss current clinical immunotherapy approaches based on TILs as well as promising directions for future expansion, and highlight existing clinical data supporting its use.

The ubiquitin system is important for drug discovery, and the discovery of selective small-molecule inhibitors of deubiquitinating enzymes (DUBs) remains an active yet extremely challenging task. With a few exceptions, previously developed inhibitors have been found to bind the evolutionarily conserved catalytic centers of DUBs, resulting in poor selectivity. The small molecule IU1 was the first-ever specific inhibitor identified and exhibited surprisingly excellent selectivity for USP14 over other DUBs. However, the molecular mechanism for this selectivity was elusive. Herein, we report the high-resolution co-crystal structures of the catalytic domain of USP14 bound to IU1 and three IU1 derivatives. All the structures of these complexes indicate that IU1 and its analogs bind to a previously unknown steric binding site in USP14, thus blocking the access of the C-terminus of ubiquitin to the active site of USP14 and abrogating USP14 activity. Importantly, this steric site in USP14 is very unique, as suggested by structural alignments of USP14 with several known DUB X-ray structures. These results, in conjunction with biochemical characterization, indicate a coherent steric blockade mechanism for USP14 inhibition by compounds of the IU series. In light of the recent report of steric blockade of USP7 by FT671, this work suggests a potential generally applicable allosteric mechanism for the regulation of DUBs via steric blockade, as showcased by our discovery of IU1-248 which is 10-fold more potent than IU1.

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1’ pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.

Nitrogen–fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N₂) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the “nitrogen problem”. Azotobacter vinelandii is a well–established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix nitrogen. In Azotobacter vinelandii, the NifA–NifL system fine–tunes the nif gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen–fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen–fixing regulation.

Post-translational modifications such as ubiquitination play important regulatory roles in several biological processes in eukaryotes. This process could be reversed by deubiquitinating enzymes (DUBs), which remove conjugated ubiquitin molecules from target substrates. Owing to their role as essential enzymes in regulating all ubiquitin-related processes, the abundance, localization, and catalytic activity of DUBs are tightly regulated. Dysregulation of DUBs can cause dramatic physiological consequences and a variety of disorders such as cancer, and neurodegenerative and inflammatory diseases. Multiple factors, such as transcription and translation of associated genes, and the presence of accessory domains, binding proteins, and inhibitors have been implicated in several aspects of DUB regulation. Beyond this level of regulation, emerging studies show that the function of DUBs can be regulated by a variety of post-translational modifications, which significantly affect the abundance, localization, and catalytic activity of DUBs. The most extensively studied post-translational modification of DUBs is phosphorylation. Besides phosphorylation, ubiquitination, SUMOylation, acetylation, oxidation, and hydroxylation are also reported in DUBs. In this review, we summarize the current knowledge on the regulatory effects of post-translational modifications of DUBs.