Differential Oligomerization of the Deubiquitinases USP25 and USP28 Regulates Their Activities.

Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.

Structural basis for the bi-specificity of USP25 and USP28 inhibitors.

The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets.

[Initial preclinical assessment on-site]. / Präklinische Ersteinschätzung am Einsatzort.

Emergency personnel assess their patients as an essential part of their daily work in emergency settings. The clinical assessment can help differentiate between immediately life-threatening conditions and milder diseases in patients. This assessment is the basis for an adequate preclinical treatment and for a rational referral to the hospital providing further care. Carried out in a thoughtful manner it allows reasonable allocation of resources for inpatient care.Well-founded training, clinical experience, scores, and tools in the form of acronyms for assessing the patient lead to successful assessments according to quality standards.The preclinical initial assessment is crucial for ensuring appropriate care for patients on site with life-threatening conditions as well as those with minor problems. Prioritization of a treatment depends on the severity of the condition or injury.

In TFIIH, XPD helicase is exclusively devoted to DNA repair.

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

XPD stalled on cross-linked DNA provides insight into damage verification.

The superfamily 2 helicase XPD is a central component of the general transcription factor II H (TFIIH), which is essential for transcription and nucleotide excision DNA repair (NER). Within these two processes, the helicase function of XPD is vital for NER but not for transcription initiation, where XPD acts only as a scaffold for other factors. Using cryo-EM, we deciphered one of the most enigmatic steps in XPD helicase action: the active separation of double-stranded DNA (dsDNA) and its stalling upon approaching a DNA interstrand cross-link, a highly toxic form of DNA damage. The structure shows how dsDNA is separated and reveals a highly unusual involvement of the Arch domain in active dsDNA separation. Combined with mutagenesis and biochemical analyses, we identified distinct functional regions important for helicase activity. Surprisingly, those areas also affect core TFIIH translocase activity, revealing a yet unencountered function of XPD within the TFIIH scaffold. In summary, our data provide a universal basis for NER bubble formation, XPD damage verification and XPG incision.

Molecular basis of the head-to-tail assembly of giant muscle proteins obscurin-like 1 and titin.

Large filament proteins in muscle sarcomeres comprise many immunoglobulin-like domains that provide a molecular platform for self-assembly and interactions with heterologous protein partners. We have unravelled the molecular basis for the head-to-tail interaction of the carboxyl terminus of titin and the amino-terminus of obscurin-like-1 by X-ray crystallography. The binary complex is formed by a parallel intermolecular beta-sheet that presents a novel immunoglobulin-like domain-mediated assembly mechanism in muscle filament proteins. Complementary binding data show that the assembly is entropy-driven rather than dominated data by specific polar interactions. The assembly observed leads to a V-shaped zipper-like arrangement of the two filament proteins.

In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair.

The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.

Al-Ansab and the Dead Sea: Mid-MIS 3 archaeology and environment of the early Ahmarian population of the Levantine corridor.

Our field data from the Upper Palaeolithic site of Al-Ansab 1 (Jordan) and from a pollen sequence in the Dead Sea elucidate the role that changing Steppe landscapes played in facilitating anatomically modern human populations to enter a major expansion and consolidation phase, known as the "Early Ahmarian", several millennia subsequent to their initial Marine Isotope Stage 4/3 migration from Africa, into the Middle East. The Early Ahmarian techno-cultural unit covers a time range between 45 ka-37 ka BP. With so far more than 50 sites found, the Early Ahmarian is the first fully Upper Palaeolithic techno-cultural unit exclusively and undisputedly related to anatomically modern human populations. In order to better understand the potentially attractive features of the Early Ahmarian environmental context that supported its persistence for over 8,000 years, we carried out a decennial research program in Jordan and in the Dead Sea. This included (1) a geoscientific and archaeological survey program in the Wadi Sabra (Jordan) with a particular focus on excavations at the Early Ahmarian site of Al-Ansab 1 alongside the detailed analysis of Quaternary sediments from the same area and (2) palaeobotanical research based on Quaternary lake deposits from the Dead Sea. Our pollen data from the Dead Sea indicate slow, low frequency vegetational variation with expanding Artemisia steppe, from 60 to 20 ka BP (MIS 3-2). Here, we see a reciprocal assimilation of southern and northern Levantine vegetation zones thereby enhancing a long-lasting south-to-north steppe corridor. The same integration process accelerated about 40 ka ago, when forested areas retreated in the Lebanese Mountains. The process then extended to encompass an area from Southern Lebanon to the Sinai Peninsula. We argue that, at the same time, the carriers of the Early Ahmarian techno-cultural unit extended their habitat from their original Mediterranean biome (in the North) to the Saharo-Arabian biome (to the South). Our excavation of Al-Ansab 1, a campsite at the eastern margins of the Early Ahmarian settlement area, indicates far reaching annual movements of small, highly mobile hunter-gatherer groups. We assume a low degree of settlement complexity, still allowing for habitat extension of the Early Ahmarian into the margins of the Levantine corridor. Due to our radiometric dates, our combined archaeological and environmental record sheds light on an evolved phase of the Early Ahmarian, around 38 ka ago, rather than the starting phase of this techno-cultural unit. Possible application of our model to the starting phase of the Early Ahmarian remains an aspect of future research.

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search.

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.

Structural diversity in the atomic resolution 3D fingerprint of the titin M-band segment.

In striated muscles, molecular filaments are largely composed of long protein chains with extensive arrays of identically folded domains, referred to as "beads-on-a-string". It remains a largely unresolved question how these domains have developed a unique molecular profile such that each carries out a distinct function without false-positive readout. This study focuses on the M-band segment of the sarcomeric protein titin, which comprises ten identically folded immunoglobulin domains. Comparative analysis of high-resolution structures of six of these domains ‒ M1, M3, M4, M5, M7, and M10 ‒ reveals considerable structural diversity within three distinct loops and a non-conserved pattern of exposed cysteines. Our data allow to structurally interpreting distinct pathological readouts that result from titinopathy-associated variants. Our findings support general principles that could be used to identify individual structural/functional profiles of hundreds of identically folded protein domains within the sarcomere and other densely crowded cellular environments.

Structural Basis of Substrate Recognition and Covalent Inhibition of Cdu1 from Chlamydia trachomatis.

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into the substrate recognition of Cdu1, active-site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong basis for future structure-guided medicinal chemistry optimization of this cyanopyrimidine scaffold into more potent and selective Cdu1 inhibitors.

The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism.

RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members.

Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense.

Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.

Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling.

The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.