The rockefeller university

Replicative helicases need loader proteins to assemble at DNA replication origins. Multiple copies of the bacteriophage lambda P (P) loader bind and load the Escherichia coli DnaB (B) replicative helicase onto single-stranded (ss) DNA from the replication origin. We find that the E. coli DnaB center dot lambda P complex exists in two forms: B6P5 and B6P6. In the 2.66 & Aring; cryo-EM structure of B6P5, five lambda P loader copies form a crown-like shape that tightly grips DnaB. In this complex, the closed, planar DnaB is reconfigured into an open spiral with a large enough breach to allow ssDNA to enter an internal chamber. Transition to the open spiral involves lambda P-induced changes to the Docking Helix (DH)-Linker Helix (LH) interface. Unexpectedly, one lambda P chain in B6P5 is positioned across the breach. The disposition of this lambda P chain implies a complex pathway for entry of a replication-origin-derived ssDNA "bubble" ssDNA into the B6P5 complex. We propose that the B6P6 complex is an early intermediate in helicase activation in which neither DnaB nor lambda P has reached its final form. In this complex, DnaB adopts a partially open, ajar planar configuration. lambda P in B6P6 interacts more loosely with DnaB. The ssDNA- and ATP-binding sites in both complexes are not correctly configured for binding or hydrolysis. Our findings detail the distinct conformations of B6P6 and B6P5, allowing us to propose a structural model for the transition from an ajar planar to an open spiral configuration in the helicase loading pathway.

Nucleoside-modified messenger RNA (mRNA) vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cell differentiation. The lipid nanoparticles (LNPs) of mRNA vaccines possess inherent adjuvant activity. However, the extent to which the nucleoside-modified mRNA is sensed and contributes to Tfh cell responses remains undefined. Herein, we deconvolute the signals induced by LNPs and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrate that the mRNA drives the production of type I interferons, which act on DCs to enhance their maturation and Tfh cell differentiation, and favors plasma cells and memory B cell responses. In parallel, LNPs, which allow for mRNA uptake by DCs within the draining lymph node, also modulate Tfh cell responses by shaping the localization of CD25+ DCs. Our work unravels distinct adjuvant features of mRNA and LNPs necessary for the induction of Tfh cells, with implications for rational vaccine design.

Replicative senescence is a powerful tumor suppressor pathway that curbs proliferation of human cells when a few critically-short telomeres activate the DNA damage response (DDR). We show that ATM is the sole DDR kinase responsible for the induction and maintenance of replicative senescence and that ATM inhibition can induce normal cell divisions in senescent cells. Compared to non-physiological atmospheric (similar to 20%) oxygen, primary fibroblast cells grown at physiological (3%) oxygen were more tolerant to critically short telomeres, explaining their extended replicative lifespan. We show that this tolerance is due to attenuation of the ATM response to double-strand breaks (DSBs) and unprotected telomeres. Our data indicate that the reduced ATM response to DSBs at 3% oxygen is due to increased ROS, which induces disulfide crosslinked ATM dimers that do not respond to DSBs. This regulation of cellular lifespan through attenuation of ATM at physiological oxygen has implications for tumor suppression through telomere shortening.

'Intrinsic immunity' is often used to refer to mechanisms of host defense operating in nonleukocytic cells. This term can refer to the intrinsic capacity of an individual cell to fend off invading microbes without help from other cells or of a group of similar cells to fend off invading microbes without help from other cell types. The intrinsic capacity of individual cells to defend themselves against invading microbes without assistance has received little attention and is the topic of this review. We also focus on nonleukocytic cells and on humans, the only species in which intrinsic immunity has been shown by genetic means to be essential for homeostasis in natural conditions at wholebody level. We review recent progress in our understanding of the type I interferon-independent intrinsic immunity of individual nonleukocytic cells, as inferred from human inborn errors of intrinsic immunity manifesting as infection or autoinflammation.

The recent Society for Molecular Biology and Evolution Satellite Meeting on De Novo Gene Birth, hosted at Texas A&M University on November 6 to 9, 2023, represented the first-ever opportunity for scientists studying the evolution and biology of de novo genes to gather through a dedicated meeting and discuss about groundbreaking discoveries in this emerging and exciting field of gene evolution. In this perspective, we discuss recent advances and major open questions in de novo gene emergence and evolution that were presented at the SMBE satellite meeting, as well as some of the key recent findings published before or since the conference. These key themes include de novo gene identification, function, and evolution, what we are learning about de novo genes from experimental analyses of random peptides, de novo gene birth and microproteins, and the role of de novo genes in human disease.

Pseudomonas aeruginosa is a leading cause of nosocomial infections, including pneumonia and urinary tract infections, and the primary cause of morbidity and mortality in cystic fibrosis patients. The emergence of multidrug-resistant strains makes these infections life-threatening. To overcome this challenge, lysocins can be employed as novel antipseudomonals. Lysocins use components of the pyocin antimicrobial system to deliver bacteriophage lysins to their peptidoglycan substrate in Pseudomonas. Peptidoglycan cleavage causes membrane destabilization, cytoplasmic leakage, and disruption of the proton motive force, thereby killing the cell. In our previous proof-of-concept study, the PyS2-GN4 lysocin killed only one-third of P. aeruginosa strains due to the targeted receptor. This limitation can now be circumvented by engineering second-generation lysocins that bind and translocate through highly conserved Pseudomonas-specific receptors. One lysocin, PyS5-I-GN4, uses a single domain from pyocin S5 to deliver the GN4 lysin through the conserved ferric pyochelin transporter, consequently killing 95% of multidrug-resistant clinical isolates tested. Importantly, PyS5-I-GN4 displayed antibiofilm properties and was bactericidal in serum and lung surfactant. Serum inactivation observed for lysins is not seen for lysocins, making this approach more effective for treating systemic Gram-negative bacterial infections. Despite its broadened pseudomonal strain coverage, PyS5-I-GN4 demonstrated narrow-spectrum antibacterial activity toward P. aeruginosa only and lacked cytotoxicity toward human cells. A single dose of lysocin was protective and reduced bacteria multiple log10-fold in the lungs and secondary organs in a neutropenic murine lung infection model. These findings support lysocins as therapeutics for P. aeruginosa and provide insight into designing future constructs for other Gram-negative pathogens.

Eukaryotic ribosomal small subunit (SSU) assembly requires the SSU processome, a nucleolar precursor containing the RNA chaperone U3 small nucleolar RNA (snoRNA). The underlying molecular mechanisms of SSU processome maturation, remodelling, disassembly and RNA quality control, and the transitions between states remain unknown owing to a paucity of intermediates1, 2-3. Here we report 16 native SSU processome structures alongside genetic data, revealing how two helicases, the Mtr4-exosome and Dhr1, are controlled for accurate and unidirectional ribosome biogenesis. Our data show how irreversible pre-ribosomal RNA degradation by the redundantly tethered RNA exosome couples the transformation of the SSU processome into a pre-40S particle, during which Utp14 can probe evolving surfaces, ultimately positioning and activating Dhr1 to unwind the U3 snoRNA and initiate nucleolar pre-40S release. This study highlights a paradigm for large dynamic RNA-protein complexes in which irreversible RNA degradation drives compositional changes and communicates these changes to govern enzyme activity while maintaining overall quality control.

Antibodies play a crucial role in adaptive immunity. They develop as B cell receptors (BCRs): membrane-bound forms of antibodies that are expressed on the surfaces of B cells. BCRs are refined through affinity maturation, a process of somatic hypermutation (SHM) and natural selection, to improve binding to an antigen. Computational models of affinity maturation have developed from two main perspectives: molecular evolution and language modeling. The molecular evolution perspective focuses on nucleotide sequence context to describe mutation and selection; the language modeling perspective involves learning patterns from large data sets of protein sequences. In this paper, we compared models from both perspectives on their ability to predict the course of antibody affinity maturation along phylogenetic trees of BCR sequences. This included models of SHM, models of SHM combined with an estimate of selection, and protein language models. We evaluated these models for large human BCR repertoire data sets, as well as an antigen-specific mouse experiment with a pre-rearranged cognate naive antibody. We demonstrated that precise modeling of SHM, which requires the nucleotide context, provides a substantial amount of predictive power for predicting the course of affinity maturation. Notably, a simple nucleotide-based convolutional neural network modeling SHM outperformed state-of-the-art protein language models, including one trained exclusively on antibody sequences. Furthermore, incorporating estimates of selection based on a custom deep mutational scanning experiment brought only modest improvement in predictive power. To support further research, we introduce EPAM (Evaluating Predictions of Affinity Maturation), a benchmarking framework to integrate evolutionary principles with advances in language modeling, offering a road map for understanding antibody evolution and improving predictive models.

Avian influenza A virus (IAV) H5N1 is an emerging threat of human pandemic. We describe a 71-year-old man who died of H5N1 pneumonia in Louisiana and whose blood contained autoantibodies neutralizing type I IFNs (AAN-I-IFNs), including the 12 IFN-alpha subtypes (1-10 ng/ml) and IFN-omega (100 pg/ml). Causality between these AAN-I-IFN and lethal outcome of avian influenza in this patient is based on (1) our previous report that AA-I-IFN underlie about 5% of cases of critical pneumonia triggered by seasonal influenza viruses in three cohorts, (2) the rarity of this combination of AAN-I-FNs in individuals over 70 years old (<1%), and (3) the rarity of lethal avian influenza among infected individuals (<1%). AAN-I-IFNs underlie a growing number of severe viral diseases, from arboviral encephalitis to viral pneumonia, particularly in the elderly. This case suggests they can also underlie life-threatening avian H5N1 influenza. The presence of AAN-I-IFN may facilitate infection, replication, and adaptation of zoonotic IAVs to humans and, therefore, human-to-human transmission.

The dominant approach to the study of living systems in the 20th century into today has been that of a reductionist approach focused on genetics and biochemistry. The hunt for genes and the elucidation of their biochemical outputs has organized funding in research, educational curricula, academic promotion, and the distribution of prestige through awards. Such reductionism has gone hand in hand with an ontology of the machine. We will discuss how viewing life as if it emanated from a set of molecular machines is the main bottleneck in addressing key questions in biology. We will discuss how moving beyond it is not contingent on new technologies but rather a refreshed perspective of life that can be termed "organic". Furthermore, we suggest that the study of how form arises, morphogenesis, is the key to an organic renewal of biology and biomedicine. Although morphogenesis is currently seen as a subsidiary branch of developmental biology as well as the consequence of molecular patterning processes at the subcellular scale, we will argue that morphology and its self-organizing capacity at the supracellular scale is the fundamental nexus in embryonic development as well as disease. We see the inability to appreciate form through an organic supracellular perspective as the principal bottleneck for making inroads into health issues such as cancer and the chronic disease epidemic.