Structural transitions modulate the chaperone activities of Grp94.

A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.

Understanding chaperone specificity: evidence for a 'client code'.

The interactions of molecular chaperones with clients can be regulated by chaperone post-translational modification (PTMs) collectively known as the 'chaperone code'. What is less understood is how PTMs on client proteins may impact chaperone-client interactions. In this forum, we discuss the possibility of a 'client code'.

CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression.

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.

Feeling the heat: how chaperones deal with biomolecular condensates.

Yoo et al. have uncovered the minimal requirements of chaperone-mediated dispersal of Pab1 biomolecular condensates. These studies expand our understanding of the uniqueness of co-chaperones and add to our fundamental understanding of the heat shock response in cells.

Biosynthesis of peptide-nucleobase hybrids in ribosomal peptides.

The main biopolymers in nature are oligonucleotides and polypeptides. However, naturally occurring peptide-nucleobase hybrids are rare. Here we report the characterization of the founding member of a class of peptide-nucleobase hybrid natural products with a pyrimidone motif from a widely distributed ribosomally synthesized and post-translationally modified (RiPP) biosynthetic pathway. This pathway features two steps where a heteromeric RRE-YcaO-dehydrogenase complex catalyzes the formation of a six-membered pyrimidone ring from an asparagine residue on the precursor peptide, and an acyl esterase selectively recognizes this moiety to cleave the C-terminal follower peptide. Mechanistic studies reveal that the pyrimidone formation occurs in a substrate-assisted catalysis manner, requiring a His residue in the precursor to activate asparagine for heterocyclization. Our study expands the chemotypes of RiPP natural products and the catalytic scope of YcaO enzymes. This discovery opens avenues to create artificial biohybrid molecules that resemble both peptide and nucleobase, a modality of growing interest.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase.

The Hsp70 family of molecular chaperones is well-conserved and expressed in all organisms. In budding yeast, cells express four highly similar cytosolic Hsp70s Ssa1, 2, 3 and 4 which arose from gene duplication. Ssa1 and 2 are constitutively expressed while Ssa3 and 4 are induced upon heat shock. Recent evidence suggests that despite their amino acid similarity, these Ssas have unique roles in the cell. Here we examine the relative importance of Ssa1-4 in the regulation of the enzyme ribonucleotide reductase (RNR). We demonstrate that cells expressing either Ssa3 or Ssa4 as their sole Ssa are compromised for their resistance to DNA damaging agents and activation of DNA damage response (DDR)-regulated transcription. In addition, we show that the steady state levels and stability of RNR small subunits Rnr2 and Rnr4 are reduced in Ssa3 or Ssa4-expressing cells, a result of decreased Ssa-RNR interaction. Interaction between the Hsp70 co-chaperone Ydj1 and RNR is correspondingly decreased in cells only expressing Ssa3 and 4. Through studies of Ssa2/4 domain swap chimeras, we determined that the C-terminal domain of Ssas are the source of this functional specificity. Taking together, our work suggests a distinct role for Ssa paralogs in regulating DNA replication mediated by C-terminus sequence variation.

Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to regulate TORC1 signaling.

The sorting nexins (SNX), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-BAR proteins, a SNX subfamily characterized by a C-terminal dimeric Bin/Amphiphysin/Rvs (BAR) lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BARs proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endo-vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and has physical and genetic interactions with the SEA complex to regulate TORC1 inactivation. We found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during TORC1 signaling during autophagy induction. Additionally, we found the SEACIT complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant endosomal TORC1 activity and subsequent Atg13 hyperphosphorylation. We have also discovered that the vacuolar localization of Vps501 is dependent upon a direct interaction with Sea1 and a unique lipid binding specificity that is also required for its function. This article is protected by copyright. All rights reserved.

Dealing with difficult clients via personalized chaperone inhibitors.

The importance of molecular chaperones in cancer is well established, yet several chaperone inhibitors have failed in clinical trials due to toxicity. Recent efforts have focused on targeting chaperone function in cancer by either manipulating the "chaperone code" or inhibiting helper cochaperones, such as DNAJA1. Tong et al. identify a novel inhibitor that specifically disrupts DNAJA1's interaction with p53, promoting p53 degradation. This finding highlights specific DNAJA1 interactions with the potential for less toxicity compared to traditional chaperone inhibitors.

The bottromycin epimerase BotH defines a group of atypical α/ß-hydrolase-fold enzymes.

D-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of L-amino acids into their D-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a D-aspartate (D-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/ß-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of L-Asp to D-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH-substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.

Author Correction: The bottromycin epimerase BotH defines a group of atypical α/ß-hydrolase-fold enzymes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cracking the Chaperone Code: Cellular Roles for Hsp70 Phosphorylation.

Heat shock protein 70 (Hsp70) is a molecular chaperone required for protein folding, cell viability, and cancer cell proliferation. Recent studies suggest that Hsp70 phosphorylation regulates important cellular processes, such as cell cycle progression, apoptosis, protein degradation, and resistance to anticancer therapeutics.

Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code.

Cells must be able to cope with the challenge of folding newly synthesized proteins and refolding those that have become misfolded in the context of a crowded cytosol. One such coping mechanism that has appeared during evolution is the expression of well-conserved molecular chaperones, such as those that are part of the heat shock protein 70 (Hsp70) family of proteins that bind and fold a large proportion of the proteome. Although Hsp70 family chaperones have been extensively examined for the last 50 years, most studies have focused on regulation of Hsp70 activities by altered transcription, co-chaperone "helper" proteins, and ATP binding and hydrolysis. The rise of modern proteomics has uncovered a vast array of post-translational modifications (PTMs) on Hsp70 family proteins that include phosphorylation, acetylation, ubiquitination, AMPylation, and ADP-ribosylation. Similarly to the pattern of histone modifications, the histone code, this complex pattern of chaperone PTMs is now known as the "chaperone code." In this review, we discuss the history of the Hsp70 chaperone code, its currently understood regulation and functions, and thoughts on what the future of research into the chaperone code may entail.

Bottromycins - biosynthesis, synthesis and activity.

Covering: 1950s up to the end of 2020Bottromycins are a class of macrocyclic peptide natural products that are produced by several Streptomyces species and possess promising antibacterial activity against clinically relevant multidrug-resistant pathogens. They belong to the ribosomally synthesised and post-translationally modified peptide (RiPP) superfamily of natural products. The structure contains a unique four-amino acid macrocycle formed via a rare amidine linkage, C-methylation and a D-amino acid. This review covers all aspects of bottromycin research with a focus on recent years (2009-2020), in which major advances in total synthesis and understanding of bottromycin biosynthesis were achieved.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

The Hsp70 co-chaperone Ydj1/HDJ2 regulates ribonucleotide reductase activity.

Hsp70 is a well-conserved molecular chaperone involved in the folding, stabilization, and eventual degradation of many "client" proteins. Hsp70 is regulated by a suite of co-chaperone molecules that assist in Hsp70-client interaction and stimulate the intrinsic ATPase activity of Hsp70. While previous studies have shown the anticancer target ribonucleotide reductase (RNR) is a client of Hsp70, the regulatory co-chaperones involved remain to be determined. To identify co-chaperone(s) involved in RNR activity, 28 yeast co-chaperone knockout mutants were screened for sensitivity to the RNR-perturbing agent Hydroxyurea. Ydj1, an important cytoplasmic Hsp70 co-chaperone was identified to be required for growth on HU. Ydj1 bound the RNR subunit Rnr2 and cells lacking Ydj1 showed a destabilized RNR complex. Suggesting broad conservation from yeast to human, HDJ2 binds R2B and regulates RNR stability in human cells. Perturbation of the Ssa1-Ydj1 interaction through mutation or Hsp70-HDJ2 via the small molecule 116-9e compromised RNR function, suggesting chaperone dependence of this novel role. Mammalian cells lacking HDJ2 were significantly more sensitive to RNR inhibiting drugs such as hydroxyurea, gemcitabine and triapine. Taken together, this work suggests a novel anticancer strategy-inhibition of RNR by targeting Hsp70 co-chaperone function.

The intracellular DNA sensors cGAS and IFI16 do not mediate effective antiviral immune responses to HSV-1 in human microglial cells.

Glia play a key role in immunosurveillance within the central nervous system (CNS) and can recognize a wide range of pathogen-associated molecular patterns (PAMPS) via members of multiple pattern recognition receptor (PRR) families. Of these, the expression of cytosolic/nuclear RNA and DNA sensors by glial cells is of particular interest as their ability to interact with intracellular nucleic acids suggests a critical role in the detection of viral pathogens. The recently discovered DNA sensors cyclic GMP-AMP synthase (cGAS) and interferon gamma-inducible protein 16 (IFI16) have been reported to be important for the recognition of DNA pathogens such as herpes simplex virus-1 (HSV-1) in peripheral human cell types, and we have recently demonstrated that human glia express cGAS and its downstream adaptor molecule stimulator of interferon genes (STING). Here, we have demonstrated that human microglial cells functionally express cGAS and exhibit robust constitutive IFI16 expression. While cGAS serves as a significant component in IRF3 activation and IFN-ß production by human microglial cells in response to foreign intracellular DNA, IFI16 is not required for such responses. Surprisingly, neither of these sensors mediate effective antiviral responses to HSV-1 in microglia, and this may be due, at least in part, to viral suppression of cGAS and/or IFI16 expression. As such, this ability may represent an important HSV immune evasion strategy in glial cells, and approaches that mitigate such suppression might represent a novel strategy to limit HSV-1-associated neuropathology.

Teicoplanin biosynthesis: unraveling the interplay of structural, regulatory, and resistance genes.

Teicoplanin (Tcp) is a clinically relevant glycopeptide antibiotic (GPA) that is produced by the actinobacterium Actinoplanes teichomyceticus. Tcp is a front-line therapy for treating severe infections caused by multidrug-resistant Gram-positive pathogens in adults and infants. In this review, we provide a detailed overview of how Tcp is produced by A. teichomyceticus by describing Tcp biosynthesis, regulation, and resistance. We summarize the knowledge gained from in vivo and in vitro studies to provide an integrated model of teicoplanin biosynthesis. Then, we discuss genetic and nutritional factors that contribute to the regulation of teicoplanin biosynthesis, focusing on those that have been successfully applied for improving teicoplanin production. A current view on teicoplanin self-resistance mechanisms in A. teichomyceticus is given, and we compare the Tcp biosynthetic gene cluster with other glycopeptide gene clusters from actinoplanetes and from unidentified isolates/metagenomics samples. Finally, we provide an outlook for further directions in studying Tcp biosynthesis and regulation.