Markus Kaiser

HTRA proteases: regulated proteolysis in protein quality control.

Controlled proteolysis underlies a vast diversity of protective and regulatory processes that are of key importance to cell fate. The unique molecular architecture of the widely conserved high temperature requirement A (HTRA) proteases has evolved to mediate critical aspects of ATP-independent protein quality control. The simple combination of a classic Ser protease domain and a carboxy-terminal peptide-binding domain produces cellular factors of remarkable structural and functional plasticity that allow cells to rapidly respond to the presence of misfolded or mislocalized polypeptides.

A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism

Pathogenic bacteria often use effector molecules to increase virulence. In most cases, the mode of action of effectors remains unknown. Strains of Pseudomonas syringae pv. syringae (Pss) secrete syringolin A (SylA), a product of a mixed non-ribosomal peptide/polyketide synthetase, in planta. Here we identify SylA as a virulence factor because a SylA-negative mutant in Pss strain B728a obtained by gene disruption was markedly less virulent on its host, Phaseolus vulgaris (bean). We show that SylA irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, thus adding proteasome inhibition to the repertoire of modes of action of virulence factors. The crystal structure of the yeast proteasome in complex with SylA revealed a novel mechanism of covalent binding to the catalytic subunits. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales, for which we demonstrate a similar proteasome inhibition mechanism. As proteasome inhibitors are a promising class of anti-tumour agents, the discovery of a novel family of inhibitory natural products, which we refer to as syrbactins, may also have implications for the development of anti-cancer drugs. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

Biology-inspired synthesis of compound libraries

Abstract.Biologically active small molecules represent the basis for chemical biology applications in which small molecules are used as chemical tools to probe biological processes. In this report, we review two approaches to design and synthesize compound libraries for biological screenings, i.e., diversity-oriented synthesis (DOS) and biology-oriented synthesis (BIOS).

Small‐Molecule Stabilization of Protein–Protein Interactions: An Underestimated Concept in Drug Discovery?

The modulation of protein-protein interactions (PPIs) has been recognized as one of the most challenging tasks in drug discovery. While their systematic development has long been considered as intractable, this view has changed over the last years, with the first drug candidates undergoing clinical studies. To date, the vast majority of PPI modulators are interaction inhibitors. However, in many biological contexts a prolonged lifespan of a PPI might be desirable, calling for the complementary approach of PPI stabilization. In fact, nature offers impressive examples of this concept and some PPI-stabilizing natural products have already found application as important drugs. Moreover, directed small-molecule stabilization has recently been demonstrated. Therefore, it is time to take a closer look at the constructive side of modulating PPIs.

Protein Quality Control in the Bacterial Periplasm

Protein quality control involves sensing and treatment of defective or incomplete protein structures. Misfolded or mislocalized proteins trigger dedicated signal transduction cascades that upregulate the production of protein quality-control factors. Corresponding proteases and chaperones either degrade or repair damaged proteins, thereby reducing the level of aggregation-prone molecules. Because the periplasm of gram-negative bacteria is particularly exposed to environmental changes and respective protein-folding stresses connected with the presence of detergents, low or high osmolarity of the medium, elevated temperatures, and the hosts immune response, fine-tuned protein quality control systems are essential for survival under these unfavorable conditions. This review discusses recent advances in the identification and characterization of the key cellular factors and the emerging general principles of the underlying molecular mechanisms.

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B Encode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis thaliana

This article characterizes two anther-expressed type III polyketide synthases that are related to chalcone synthase. The results support the hypothesis that the enzymes are involved in an ancient sporopollenin biosynthetic pathway that catalyzes sequential modification of fatty acid starter molecules to generate alkyl α-pyrone polyketide sporopollenin components of the pollen exine. Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a Ki′ of 8.65 ± 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics.

|[beta]|-Lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana

New activity-based probes are essential for expanding studies on the hundreds of serine and cysteine proteases encoded by the genome of Arabidopsis thaliana. To monitor protease activities in plant extracts, we generated biotinylated peptides containing a beta-lactone reactive group. These probes cause strong labeling in leaf proteomes. Unexpectedly, labeling was detected at the N terminus of PsbP, nonproteolytic protein of photosystem II. Inhibitor studies and reverse genetics led to the discovery that this unusual modification is mediated by a single plant-specific, papain-like protease called RD21. In cellular extracts, RD21 accepts both beta-lactone probes and peptides as donor molecules and ligates them, probably through a thioester intermediate, to unmodified N termini of acceptor proteins.

Substrate-induced remodeling of the active site regulates human HTRA1 activity.

Crystal structures of active and inactive conformations of the human serine protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other serine proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features.

The Core Structure of TMC-95A Is a Promising Lead for Reversible Proteasome Inhibition

Correspondingly,great attention has recently been paid to the discovery ofpotentandselectiveproteasomeinhibitorsbystructure-baseddesignornaturalproductscreeningapproaches.Mostofthesynthetic inhibitors consisting of peptide aldehydes, boro-nates, and vinylsulfones, as well as the natural productslactacystinandepoxymicinsinhibitinamoreorlessselectivemanner the proteasome by reaction with the N-terminalthreonineresidue(forarecentreviewseeref.[5]).Anotableexceptionisthehighlyselectiveandcompetitiveproteasomeinhibitor TMC-95A, which was isolated from the fermenta-tionbrothofApiosporamontagneiSacc.TC1093.