Site-directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA-A1/A36-specific monoclonal antibody.

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5 Å radius. Eplet 44KM has been previously redefined to the antibody-verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody-induction and binding are limited. In this proof-of-concept study, we performed site-directed mutagenesis to narrow down the antibody-verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12.

Imaging findings in invasive rhino-orbito-cerebral mucormycosis in post-COVID-19 patients.

Rhino-orbito-cerebral mucormycosis (ROCM) is a life-threatening addition to the COVID-19 disease spectrum and is caused by an angioinvasive saprophytic opportunistic fungus. Early diagnosis is important to avoid disease spread and mortality. Contrast-enhanced magnetic resonance imaging plays a major role in detection of intraorbital and intracranial extension. We present imaging findings of 15 patients with post-COVID-19 rhino-orbito-cerebral mucormycosis who were diagnosed with invasive sinus mucormycosis at our institution and are currently undergoing treatment. All patients were diabetics, and 80% had a history of steroid intake during the course of COVID-19 treatment. There was a male preponderance (73.3%). The maxillary sinus was most commonly involved (86.7%). Orbital and intracranial invasion was seen in 73.3% and 60% of patients, respectively. The presence of retroantral, facial, infratemporal, and orbital fat stranding was an early sign of extrasinus spread. Other common sites of extrasinus involvement were the orbit and face, followed by the orbital apex, masticator space, pterygopalatine fossa, bone, skull base, cavernous sinus, brain, and internal carotid artery. In conclusion, early detection of extrasinus spread of mucormycosis by imaging is important so that aggressive treatment can be given and mortality can be reduced.

Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones.

Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat ß-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

Mak5 and Ebp2 act together on early pre-60S particles and their reduced functionality bypasses the requirement for the essential pre-60S factor Nsa1.

Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these 'Mak5 cluster' factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation.