Miguel Santos-Fernandez

Florida International University

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Francisco Fernandez‐Lima

Florida International University

Kévin Jeanne Dit Fouque

Florida International University

Mario Gomez-Hernandez

Texas A&M University

Wilfred A. van der Donk

Howard Hughes Medical Institute

Julian D. Hegemann

Helmholtz Institute for Pharmaceutical Research Saarland

Tung Le

University of Illinois Urbana-Champaign

Jeremy W. Chambers

Florida International University

Claudio D. Navo

CIC bioGUNE

Katlyn K. Meier

University of Miami

Desmond A. Kaplan

AndroScience (United States)

Cooperative Institutions

Florida International University

Howard Hughes Medical Institute

University of Illinois Urbana-Champaign

University of Miami

CIC bioGUNE

Ikerbasque

Universidad de La Rioja

Boeing (Spain)

Digital Research Alliance of Canada

Texas A&M University

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Substrate Sequence Controls Regioselectivity of Lanthionine Formation by ProcM

Journal of the American Chemical Society (2021)

Tung Le Kévin Jeanne Dit Fouque Miguel Santos-Fernandez Claudio D. Navo Gonzalo Jiménez‐Osés

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry–tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.

Lanthionine

Sequence (biology)

10.1021/jacs.1c09370

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A Bifunctional Leader Peptidase/ABC Transporter Protein Is Involved in the Maturation of the Lasso Peptide Cochonodin I from Streptococcus suis

Journal of Natural Products (2021)

Julian D. Hegemann Kévin Jeanne Dit Fouque Miguel Santos-Fernandez Francisco Fernandez‐Lima

Lasso peptides are members of the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we describe the first lasso peptide originating from a biosynthetic gene cluster belonging to a unique lasso peptide subclade defined by the presence of a bifunctional protein harboring both a leader peptidase (B2) and an ABC transporter (D) domain. Bioinformatic analysis revealed that these clusters also encode homologues of the NisR/NisK regulatory system and the NisF/NisE/NisG immunity factors, which are usually associated with the clusters of antimicrobial class I lanthipeptides, such as nisin, another distinct RiPP subfamily. The cluster enabling the heterologous production of the lasso peptide cochonodin I in E. coli originated from Streptococcus suis LSS65, and the threaded structure of cochonodin I was evidenced through extensive MS/MS analysis and stability assays. It was shown that the ABC transporter domain from SsuB2/D is not essential for lasso peptide maturation. By extensive genome mining dedicated exclusively to other lasso peptide biosynthetic gene clusters featuring bifunctional B2/D proteins, it was furthermore revealed that many bacteria associated with human or animal microbiota hold the biosynthetic potential to produce cochonodin-like lasso peptides, implying that these natural products might play roles in human and animal health.

Lantibiotics

Streptococcus suis

Gene cluster

Lasso

10.1021/acs.jnatprod.1c00514

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Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry

Analytical and Bioanalytical Chemistry (2021)

Kévin Jeanne Dit Fouque Julian D. Hegemann Miguel Santos-Fernandez Tung Le Mario Gomez-Hernandez

Thioether

Electron-capture dissociation

Ion-mobility spectrometry

Ion-mobility spectrometry–mass spectrometry

10.1007/s00216-021-03437-x

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Effective discrimination of gas-phase peptide conformers using TIMS-ECD-ToF MS/MS

Analytical Methods (2021)

Kévin Jeanne Dit Fouque Malte Wellmann Dennys Leyva Miguel Santos-Fernandez Yarixa L. Cintron-Diaz

Model peptides ( e.g. , substance P, bradykinin, angiotensin I and AT-Hook 3) were studied using ion mobility and ECD/CID fragmentation in a TIMS-q-EMS-ToF MS/MS platform.

Ion-mobility spectrometry

Conformational isomerism

Fragmentation

10.1039/d1ay01461g

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Citations (10)

Multiple Copper Ions Bind to and Promote the Oligomerization of Huntingtin Protein with Nonpathological Repeat Expansions

Biochemistry (2025)

Deepa Neupane Miguel Santos-Fernandez Francisco Fernandez‐Lima Katlyn K. Meier

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by the expression of huntingtin protein (htt) that has a polyglutamine (CAG; polyQ) repeat domain consisting of 36 or more glutamines (mhtt). Historically, mhtt is more broadly associated with HD severity, as are elevated metal levels observed in HD patients. The depletion of wild-type (WT) htt (fewer than 36Qs) is also recognized as a contributing factor to HD progression; however, many questions remain about the interactions of biorelevant metals with WT htt and the impact of the interactions on protein aggregation. In the present work, we utilize a combination of biochemical assays and spectroscopic techniques to provide insights into the interaction of copper with an in vitro htt model (N171-17Q). Herein, we use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering to show that the addition of equimolar or higher concentrations of Cu(II) to htt induces time- and temperature-dependent protein oligomerization/aggregation. Additionally, chelation assays, trapped ion mobility spectrometry, and mass spectrometry confirm the (i) rapid reduction of Cu(II) in the presence of N171-17Q htt, (ii) direct binding of multiple copper ions per protein, and (iii) complex Cu:htt speciation profile with a preference for three distinct Cu:htt states. These findings contribute to our molecular level understanding of copper's role in the depletion and oligomerization/aggregation of WT htt while underscoring the physiological significance of our work, its potential relevance to metal binding in mhtt, and its significance for identifying new avenues for biomarker exploration and therapeutic design strategies.

10.1021/acs.biochem.5c00012

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Integration of Trapped Ion Mobility Spectrometry and Ultraviolet Photodissociation in a Quadrupolar Ion Trap Mass Spectrometer

Analytical Chemistry (2023)

Miguel Santos-Fernandez Kévin Jeanne Dit Fouque Francisco Fernandez‐Lima

There is a growing demand for lower-cost, benchtop analytical instruments with complementary separation capabilities for the screening and characterization of biological samples. In this study, we report on the custom integration of trapped ion mobility spectrometry and ultraviolet photodissociation capabilities in a commercial Paul quadrupolar ion trap multistage mass spectrometer (TIMS-QIT-MSn UVPD platform). A gated TIMS operation allowed for the accumulation of ion mobility separated ion in the QIT, followed by a mass analysis (MS1 scan) or m/z isolation, followed by selected collision induced dissociation (CID) or ultraviolet photodissociation (UVPD) and a mass analysis (MS2 scan). The analytical potential of this platform for the analysis of complex and labile biological samples is illustrated for the case of positional isomers with varying PTM location of the histone H4 tryptic peptide 4-17 singly and doubly acetylated and the histone H3.1 tail (1-50) singly trimethylated. For all cases, a baseline ion mobility precursor molecular ion preseparation was obtained. The tandem CID and UVPD MS2 allowed for effective sequence confirmation as well as the identification of reporter fragment ions associated with the PTM location; a higher sequence coverage was obtained using UVPD when compared to CID. Different from previous IMS-MS implementation, the novel TIMS-QIT-MSn UVPD platform offers a lower-cost alternative for the structural characterization of biological molecules that can be widely disseminated in clinical laboratories.

Ion-mobility spectrometry

Ultraviolet

Quadrupole ion trap

Collision-induced dissociation

Electron-transfer dissociation

Characterization

10.1021/acs.analchem.3c01220

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Citations (1)

Inhibition of HMGA2 binding to AT-rich DNA by its negatively charged C-terminus

Nucleic Acids Research (2025)

Linjia Su Zifang Deng Miguel Santos-Fernandez Kévin Jeanne Dit Fouque Prem P. Chapagain

Abstract The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged ‘AT-hooks’ and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its ‘AT-hooks’ and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged ‘AT-hooks.’ In this paper, we demonstrate that the C-terminus significantly influences HMGA2’s DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95–108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2’s binding to FL814, likely by interacting with the ‘AT-hooks,’ as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP’s binding to the ‘AT-hooks.’ Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences.

C-terminus

HMG-box

10.1093/nar/gkaf035

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